Identification of rhizome-specific genes by genome-wide differential expression Analysis in Oryza longistaminata

<p>Abstract</p> <p>Background</p> <p>Rhizomatousness is a key component of perenniality of many grasses that contribute to competitiveness and invasiveness of many noxious grass weeds, but can potentially be used to develop perennial cereal crops for sustainable farmers in hilly areas of tropical Asia. <it>Oryza longistaminata</it>, a perennial wild rice with strong rhizomes, has been used as the model species for genetic and molecular dissection of rhizome development and in breeding efforts to transfer rhizome-related traits into annual rice species. In this study, an effort was taken to get insights into the genes and molecular mechanisms underlying the rhizomatous trait in <it>O. longistaminata </it>by comparative analysis of the genome-wide tissue-specific gene expression patterns of five different tissues of <it>O. longistaminata </it>using the Affymetrix GeneChip Rice Genome Array.</p> <p>Results</p> <p>A total of 2,566 tissue-specific genes were identified in five different tissues of <it>O. longistaminata</it>, including 58 and 61 unique genes that were specifically expressed in the rhizome tips (RT) and internodes (RI), respectively. In addition, 162 genes were up-regulated and 261 genes were down-regulated in RT compared to the shoot tips. Six distinct <it>cis</it>-regulatory elements (CGACG, GCCGCC, GAGAC, AACGG, CATGCA, and TAAAG) were found to be significantly more abundant in the promoter regions of genes differentially expressed in RT than in the promoter regions of genes uniformly expressed in all other tissues. Many of the RT and/or RI specifically or differentially expressed genes were located in the QTL regions associated with rhizome expression, rhizome abundance and rhizome growth-related traits in <it>O. longistaminata </it>and thus are good candidate genes for these QTLs.</p> <p>Conclusion</p> <p>The initiation and development of the rhizomatous trait in <it>O. longistaminata </it>are controlled by very complex gene networks involving several plant hormones and regulatory genes, different members of gene families showing tissue specificity and their regulated pathways. Auxin/IAA appears to act as a negative regulator in rhizome development, while GA acts as the activator in rhizome development. Co-localization of the genes specifically expressed in rhizome tips and rhizome internodes with the QTLs for rhizome traits identified a large set of candidate genes for rhizome initiation and development in rice for further confirmation.</p

Transcriptional and Metabolomic Analyses Indicate that Cell Wall Properties are Associated with Drought Tolerance in Brachypodium distachyon

Brachypodium distachyon is an established model for drought tolerance. We previously identified accessions exhibiting high tolerance, susceptibility and intermediate tolerance to drought; respectively, ABR8, KOZ1 and ABR4. Transcriptomics and metabolomic approaches were used to define tolerance mechanisms. Transcriptional analyses suggested relatively few drought responsive genes in ABR8 compared to KOZ1. Linking these to gene ontology (GO) terms indicated enrichment for “regulated stress response”, “plant cell wall” and “oxidative stress” associated genes. Further, tolerance correlated with pre-existing differences in cell wall-associated gene expression including glycoside hydrolases, pectin methylesterases, expansins and a pectin acetylesterase. Metabolomic assessments of the same samples also indicated few significant changes in ABR8 with drought. Instead, pre-existing differences in the cell wall-associated metabolites correlated with drought tolerance. Although other features, e.g., jasmonate signaling were suggested in our study, cell wall-focused events appeared to be predominant. Our data suggests two different modes through which the cell wall could confer drought tolerance: (i) An active response mode linked to stress induced changes in cell wall features, and (ii) an intrinsic mode where innate differences in cell wall composition and architecture are important. Both modes seem to contribute to ABR8 drought tolerance. Identification of the exact mechanisms through which the cell wall confers drought tolerance will be important in order to inform development of drought tolerant crops

Plant cell walls tackling climate change : insights into plant cell wall remodeling, its regulation, and biotechnological strategies to improve crop adaptations and photosynthesis in response to global warming

Plant cell wall (CW) is a complex and intricate structure that performs several functions throughout the plant life cycle. The CW of plants is critical to the maintenance of cells\u2019 structural integrity by resisting internal hydrostatic pressures, providing flexibility to support cell division and expansion during tissue differentiation, and acting as an environmental barrier that protects the cells in response to abiotic stress. Plant CW, comprised primarily of polysaccharides, represents the largest sink for photosynthetically fixed carbon, both in plants and in the biosphere. The CW structure is highly varied, not only between plant species but also among different organs, tissues, and cell types in the same organism. During the developmental processes, the main CW components, i.e., cellulose, pectins, hemicelluloses, and different types of CW-glycoproteins, interact constantly with each other and with the environment to maintain cell homeostasis. Differentiation processes are altered by positional effect and are also tightly linked to environmental changes, affecting CW both at the molecular and biochemical levels. The negative effect of climate change on the environment is multifaceted, from high temperatures, altered concentrations of greenhouse gases such as increasing CO2 in the atmosphere, soil salinity, and drought, to increasing frequency of extreme weather events taking place concomitantly, therefore, climate change affects crop productivity in multiple ways. Rising CO2 concentration in the atmosphere is expected to increase photosynthetic rates, especially at high temperatures and under water-limited conditions. This review aims to synthesize current knowledge regarding the effects of climate change on CW biogenesis and modification. We discuss specific cases in crops of interest carrying cell wall modifications that enhance tolerance to climate change-related stresses; from cereals such as rice, wheat, barley, or maize to dicots of interest such as brassica oilseed, cotton, soybean, tomato, or potato. This information could be used for the rational design of genetic engineering traits that aim to increase the stress tolerance in key crops. Future growing conditions expose plants to variable and extreme climate change factors, which negatively impact global agriculture, and therefore further research in this area is critical

Molecular studies on compatibility in the mutualistic plant root-Piriformospora indica interaction

Plants have developed diverse strategies for protection against the threat of invading pathogens. In order to improve their performance as well as to evade abiotic and biotic stresses, one strategy of plants is to establish associations with beneficial microbial organisms. Piriformospora indica is a root interacting fungus, which transfers several benefits to colonized plants like a better tolerance to various biotic and abiotic stresses, as well as an improved plant growth and yield. P. indica colonizes a broad range of monocot and dicot plants. This broad host range indicates that P. indica has developed efficient strategies to overcome innate immune responses and to manipulate the metabolism in different plants. This is even more intriguing as the fungus was shown to follow an initial biotrophic colonisation strategy at which penetrated cells are living. Plant colonizing microbes are known to secrete proteins (also called effectors) in order to modify host physiology and modulate plant defense mechanisms and, hence, confer compatibility. The aim of this study was to identify P. indica effector proteins as well as plant compatibility factors that are involved in the manipulation of those processes required for successful fungal establishment in planta. Therefore, two different strategies were followed. In the first approach, the so-called yeast signal sequence trap (YSST) assay was established. As the result of YSST, several plant genes were identified that are known to be involved in stress responses and cell wall development. These genes were shown to have a specific expression in barley roots during P. indica colonisation. In addition, a fungal gene was identified that does not show any similarities to other sequences deposited in public databases. The identified P. indica protein (PIALH43) carries a signal peptide and was shown to be induced during barley root colonisation. Interestingly, PIALH43 harbours a highly conserved C-terminal RING finger motif. In silico protein modelling of PIALH43 confirmed a 3D structural overlap and verified the accurate conformation of the E2 binding residues when compared with known human and plant ubiquitin ligases. Moreover, E3 ligase activity of PIALH43 was confirmed in vitro. Currently, PIALH43 is overexpressed in planta and in P. indica in order to study its function in mutualistic root colonisation. In a second approach, a simplified subtraction-based assay, designated Transcript Subtractive Hybridization (TSH), was established to identify and study plant compatibility factors in the barley-P. indica interaction. The subtraction assay delivered various differentially regulated genes. These genes are known to be involved in stress responses, phytohormone- and secondary metabolism, autophagy, and protein processing. Among the up-regulated candidates was a gene encoding S-adenosylmethionine synthetase 2, which is thought to be involved in the synthesis of ethylene. De novo synthesis of ethylene during root colonization was verified by quantifying the ethylene precursor 1-aminocyclopropane 1-carboxylic acid (ACC) in barley and by cytologically monitoring GUS accumulation in ACC synthase reporter plants of Arabidopsis. In addition, the effects of ethylene precursor ACC or ethylene antagonist 1-methylcyclopropene (MCP) was determined. In these pharmacological experiments, barley plants were about 40% less colonised by P. indica after application of MCP while treatment with ACC resulted in significant increase (~ 60%) in colonization. To further elucidate the impact of ethylene on plant root colonization by P. indica, genetic analyses were performed with Arabidopsis mutants altered in ethylene synthesis and signaling at early biotrophic (~ 3 dai) and later cell death-associated colonization phases (~ 14 dai). In accordance with the studies in barley, Arabidopsis mutants ctr1-1 (constitutive ethylene signaling) and eto1-1 (ethylene overproducer) exhibited a significant increase in fungal colonization (especially at later interaction stages), while a reduced colonization was observed in ein2-1 (ethylene insensitive). In summary, ethylene might function as general plant compatibility factor in the plant-P. indica system.Pflanzen haben verschieden Strategien entwickelt, um sich vor den Bedrohungen durch eindringende Pathogene zu schützen. Um ihre Leistungsfähigkeit zu verbessern und abiotischem und biotischem Stress zu entgehen, besteht eine Strategie der Pflanzen darin, Verbindungen mit nützlichen Mikroorganismen einzugehen. Piriformospora indica ist ein Pilz, der mit Pflanzenwurzeln interagiert und der besiedelten Pflanze verschiedene Vorteile verschafft, zum Beispiel eine bessere Toleranz gegenüber verschiedenen biotischen und abiotischen Stressfaktoren, verbessertes Wachstum und höhere Ernteerträge. P. indica ist in der Lage, ein breites Spektrum an monokotylen und dikotylen Pflanzen zu besiedeln. Dieses breite Wirtsspektrum deutet darauf hin, dass P. indica effiziente Mechanismen entwickelt hat, um pflanzliche Immunantworten zu überwinden und den Metabolismus verschiedener Pflanzen zu manipulieren. Dies ist umso mehr erstaunlich, da gezeigt werden konnte, dass der Pilz zu Beginn der Besiedlung in einer biotrophen Phase lebende Zellen penetriert. Pflanzen besiedelnde Mikroorganismen sekretieren bekanntermaßen Proteine (so genannte Effektoren), um die Wirtsphysiologie zu verändern, die Abwehrmechanismen der Pflanze zu modulieren und schließlich Kompatibilität zu erreichen. Das Ziel dieser Untersuchungen war es, Effektorproteine von P. indica und Kompatibilitäts-faktoren der Pflanze zu identifizieren, die an manipulativen Prozessen beteiligt sind und die erfolgreiche Etablierung des Pilzes in der Pflanze ermöglichen. Hierfür wurden zwei verschiedene Strategien verfolgt. Zunächst wurde der so genannte yeast signal sequence trap (YSST) etabliert. Als Ergebnis des YSST konnten verschiedene pflanzliche Gene identifiziert werden, die an Stressantworten und Zellwandbildung beteiligt sind. Es wurde gezeigt, dass diese Gene spezifisch in Gerstenwurzeln exprimiert wurden, wenn diese mit P. indica besiedelt waren. Zusätzlich konnte ein pilzliches Gen identifiziert werden, das keinerlei Ähnlichkeiten mit bisher bekannten Sequenzen aus öffentlich zugänglichen Datenbanken aufweist. Das entdeckte P. indica-Protein (PIALH43) trägt ein Signalpeptid, es wurde gezeigt, dass es während der Besiedlung von Gerstenwurzeln exprimiert wird. Interessanterweise beinhaltet PIALH43 ein stark konserviertes, C-terminales RING-Finger Motiv. In silico Proteinmodellierung von PIALH43 bestätigte eine dreidimensinole Überlappung und wies die genaue Konformation der der E2-Bindestellen im Vergleich mit bekannten menschlichen und pflanzlichen Ubiquitin-Ligasen nach. Die E3-Ligase-Aktivität konnte in vitro bekräftigt werden. Im Moment wird PIALH43 in der Pflanze (Arabidopsis thaliana) und in P. indica überexprimiert, um seine Funktion in der mutualistischen Wurzelkolonisierung zu studieren. In einer weiteren Annäherung wurde eine vereinfachte, auf Subtraktion basierende Methode, bezeichnet als Transcript Substractive Hybridization (TSH), etabliert. Mit Hilfe dieser Methode wurden pflanzliche Kompatibilitätsfaktoren der Gerste-P. indica Interaktion identifiziert und studiert. Die Subtraktionsmethode lieferte viele verschieden regulierte Gene. Die gefundenen Gene sind bekanntermaßen in Stressantworten, Phytohormon- und Sekundärmetabolismus, Autophagie und Proteinprozessierung beteiligt. Unter den hochregulierten Kandidaten war eine S-Adenosylmethionin-Synthetase 2, bei der vermutet wird, dass sie in der Synthese von Ethylen eine Rolle spielt. Die de novo Synthese von Ethylen während der Wurzelbesiedlung wurde durch die Quantifizierung des Ethylen-Vorläufers 1-Aminocyclopropan-1-carboxylsäure (ACC) in Gerste und durch zytologische Kontrolle der Akkumulation von GUS in ACC-Synthase-Reporterpflanzen (Arabidopsis) bestätigt. Zusätzlich wurden die Effekte des Ethylen Vorläufers ACC und des Ethylen-Antagonists 1-Methylcyclopropene (MCP) auf die Besiedlung bestimmt. In diesen pharmakologischen Untersuchungen waren Gerstenpflanzen nach der Zugabe von MCP um etwa 40 % weniger mit P. indica besiedelt, während die Behandlung mit ACC zu einem signifikanten Anstieg (~ 60 %) in der Besiedlung führte. Um den Einfluss von Ethylen auf die Besiedlung von Pflanzenwurzeln durch P. indica weiter zu beleuchten, wurden genetische Untersuchungen mit Arabidopsis-Mutanten, deren Ethylensynthese und Ethylen-Signalwege verändert waren, in der frühen biotrophen (~ 3 days after inoculation, dai) und späteren Zelltod-assoziierten Phase (~ 14 dai) durchgeführt. In Übereinstimmung mit Studien in Gerste waren die Arabidopsis-Mutanten ctr1-1 (Ethylen-Signalweg konstitutiv aktiv) und eto1-1 (Ethylen-Überproduzierer) signifikant stärker besiedelt, vor allem in späteren Interaktionsstadien. Bei der Ethylen-insensitiven Mutante ein2-1 wurde hingegen eine reduzierte Besiedlung beobachtet. Zusammenfassend scheint Ethylen ein genereller Kompatibilitätsfaktor zu sein, der durch den Pilz rekrutiert wird, um verschiedene Wirtspflanzen zu besiedeln, wie hier beispielhaft an den Modellpflanzen Gerste und Arabidopsis gezeigt werden konnte

Rice Responses to Water Limiting Conditions: Improving Stress Management by Exploiting Genetics and Physiological Processes

Water-limiting conditions can severely affect rice yield. Therefore, increasing plant tolerance to water stress is a priority for many rice breeding programs. However, improving rice tolerance to this abiotic stress comes with several complications related to the seeding practices, the adopted water management system and the growth stage where water stress occurs. For this reason, it is challenging to outline single ideotypes showing traits suitable for overcoming drought at different times during the life cycle of rice in diverse cropping ecosystems. The current knowledge of genomics and biochemicals can contribute to drawing rice ideotypes flexible towards diverse water availability conditions. Traits identified in accessions of the wild ancestor of cultivated rice, as well as other wild rice species, in Oryza glaberrima and weedy rice were demonstrated to confer enhanced tolerance to water stress, while screenings of cultivated rice germplasms identified several genes/loci improving water stress resistance. New frontiers are represented by the dissection of the epigenetic control of stress tolerance and the implementation of the contribution of favorable microbiota. Innovative breeding technologies, whose feasibility is related to advancements in genomic analyses, are contributing to enhancing the knowledge-based development of water stress-tolerant rice varieties

How an ancient, salt-tolerant fruit crop, Ficus carica L., copes with salinity: a transcriptome analysis

Although Ficus carica L. (fig) is one of the most resistant fruit tree species to salinity, no comprehensive studies are currently available on its molecular responses to salinity. Here we report a transcriptome analysis of F. carica cv. Dottato exposed to 100 mM sodium chloride for 7 weeks, where RNA-seq analysis was performed on leaf samples at 24 and 48 days after the beginning of salinization; a genomederived fig transcriptome was used as a reference. At day 24, 224 transcripts were significantly upregulated and 585 were down-regulated, while at day 48, 409 genes were activated and 285 genes were repressed. Relatively small transcriptome changes were observed after 24 days of salt treatment, showing that fig plants initially tolerate salt stress. However, after an early down-regulation of some cell functions, major transcriptome changes were observed after 48 days of salinity. Seven weeks of 100 mM NaCl dramatically changed the repertoire of expressed genes, leading to activation or reactivation of many cell functions. We also identified salt-regulated genes, some of which had not been previously reported to be involved in plant salinity responses. These genes could be potential targets for the selection of favourable genotypes, through breeding or biotechnology, to improve salt tolerance in fig or other crops

SNP characterizaiton and genetic and molecular analysis of mutants affecting fiber development in cotton

Cotton (Gossypium spp.) is the world’s leading textile fiber crop, and an important source of oil and protein. Insufficient candidate gene derived-markers suitable for genetic mapping and limited information on genes that control economically important traits are the major impediments to the genetic improvement of Upland cotton (G. hirsutum L.). The objectives of this study were to develop a SNP marker discovery strategy in tetraploid cotton species, SNP characterization and marker development from fiber initiation and elongation related genes, chromosomal assignment of these genes by SNP marker-based deletion analysis or linkage mapping, and genetic and molecular analysis of mutants affecting cotton fiber development. Phylogenetic grouping and comparision to At- and Dt-genome putative ancestral diploid species of allotetraploid cotton facilitated differentiation between genome specific polymorphisms (GSPs) and marker-suitable locus-specific polymorphisms (LSPs). By employing this strategry, a total of 222 and 108 SNPs were identified and the average frequency of SNP was 2.35% and 1.30% in six EXPANSIN A genes and six MYB genes, respectively. Both gene families showed independent and incongruent evolution in the two subgenomes and a faster evolution rate in Dt-genome than that in At-genome. SNPs were concordantly mapped to different chromsomes, which confirmed their value as candidate gene marker and indicated the reliability of SNP discovery stragey. QTL mapping by two F2 populations developed from fiber mutants detected major QTL which explain 62.8-87.1% of the phenotypic variation for lint percentage or lint index in the vicinity of BNL3482-138 on chromosome 26. Single marker regression analyses indicated STV79-108, which was located to the long arm of chromosome 12 (the known location of N1 and perhaps n2 loci), also had significant association (R2 % value 15.4-30.6) with lint percentage, lint index, embryo protein percentage and micronaire. Additional QTL and significant markers associated with other seed and fiber traits were detected on different chromosomes. Inheritance analysis indicated that both genetic models N1N1n2n2 and n2n2li3lisub\u3e3 could lead to the fiberless phenotype. The observation of fuzzless-short lint phenotype indicated fiber initiation and elongation were controlled by different mechanisms. The penetrance of Li2 gene expression was observed in this study

Generation, Annotation and Analysis of First Large-Scale Expressed Sequence Tags from Developing Fiber of Gossypium barbadense L

BACKGROUND: Cotton fiber is the world's leading natural fiber used in the manufacture of textiles. Gossypium is also the model plant in the study of polyploidization, evolution, cell elongation, cell wall development, and cellulose biosynthesis. G. barbadense L. is an ideal candidate for providing new genetic variations useful to improve fiber quality for its superior properties. However, little is known about fiber development mechanisms of G. barbadense and only a few molecular resources are available in GenBank. METHODOLOGY AND PRINCIPAL FINDINGS: In total, 10,979 high-quality expressed sequence tags (ESTs) were generated from a normalized fiber cDNA library of G. barbadense. The ESTs were clustered and assembled into 5852 unigenes, consisting of 1492 contigs and 4360 singletons. The blastx result showed 2165 unigenes with significant similarity to known genes and 2687 unigenes with significant similarity to genes of predicted proteins. Functional classification revealed that unigenes were abundant in the functions of binding, catalytic activity, and metabolic pathways of carbohydrate, amino acid, energy, and lipids. The function motif/domain-related cytoskeleton and redox homeostasis were enriched. Among the 5852 unigenes, 282 and 736 unigenes were identified as potential cell wall biosynthesis and transcription factors, respectively. Furthermore, the relationships among cotton species or between cotton and other model plant systems were analyzed. Some putative species-specific unigenes of G. barbadense were highlighted. CONCLUSIONS/SIGNIFICANCE: The ESTs generated in this study are from the first large-scale EST project for G. barbadense and significantly enhance the number of G. barbadense ESTs in public databases. This knowledge will contribute to cotton improvements by studying fiber development mechanisms of G. barbadense, establishing a breeding program using marker-assisted selection, and discovering candidate genes related to important agronomic traits of cotton through oligonucleotide array. Our work will also provide important resources for comparative genomics, polyploidization, and genome evolution among Gossypium species

GENOME-WIDE ASSOCIATION AND EXPRESSION STUDIES FOR IDENTIFICATION OF QTLS AND CANDIDATE GENES UNDERLYING ABIOTIC STRESS TOLERANCE DURING GERMINATION STAGE OF RICE

Rice is one of the most important cereal crops feeding more than half of the world’s population. Due to extreme climatic condition, different abiotic stresses like hypoxia stress and chilling stress have been the biggest threat to rice production. Direct sowing method is the most preferred way of planting in Asian countries and in U.S. due to the lower cost of planting and less labor requirement. The major challenge associated with direct sowing is flash flooding that can happen immediately after sowing due to unpredicted rainfall. In this study, we evaluated more than 250 rice accessions belonging to different groups of rice for various traits related to chilling tolerance and hypoxia tolerance during germination stage. Compressed Mixed Linear Model (CMLM) of GAPIT was used to conduct GWAS analysis for the identification of QTLs. From the GWAS study conducted for chilling stress tolerance, we identified 41 QTLs associated with different chilling indices like low temperature germinability, germination index, coleoptile growth under cold condition, plumule length at 4 d recovery, and plumule growth rate after cold germination. Out of 41 QTLs identified in the whole panel, 14 QTLs were potentially colocalized with known genes/QTLs and 27 QTLs were found to be novel. From the GWAS analysis of hypoxia stress tolerance traits, there were 24 significant SNPs identified to be associated with different traits measured under hypoxia stress. Out of the 24 significant SNPs discovered in the whole panel, 11 QTLs were found to be potentially colocalized with previously identified candidate genes underlying flooding tolerance mechanism in rice. From the phenotypic evaluation of the whole panel for chilling stress tolerance and hypoxia stress tolerance, two lines with contrasting phenotypes under each stress condition were selected and used for global gene expression analysis. The results of these transcriptomics studies have provided new insights of underlying biological processes, molecular functions and cellular components related to the phenotypic differences of the contrasting lines. The findings of our study will help in identification of promising candidate genes underlying hypoxia stress and chilling stress tolerance and would eventually assist rice breeding program to develop improved tolerant rice cultivars

Transcriptome Analysis for Abiotic Stresses in Rice (<em>Oryza sativa</em> L.)

Rice, a model monocot system, belongs to the family Poaceae and genus Oryza. Rice is the second largest produced cereal and staple food crop fulfilling the demand of half the world’s population. Though rice demand is growing exponentially, its production is severely affected by variable environmental changes. The various abiotic factors drastically reduce the rice plant growth and yield by affecting its different growth stages. To fulfill the growing demand of rice, it is imperative to understand its molecular responses during stresses and to develop new varieties to overcome the stresses. Earlier, the microarray experiments have been used for the identification of coexpressive gene networks during various conditions in crop plants. Though the microarray experiments provided very useful information, the unviability of genome-wide information did not provide complete information about the regulatory gene networks involved in the stress response. The advancement of molecular techniques provided breakthrough to understanding the complex regulatory gene networks and their signaling pathways during stresses. The high-throughput RNA sequencing data have opened the floodgate of transcriptome data in rice. Here we have summarized some of the transcriptome data for abiotic molecular responses in rice, which further help to understand their complex regulatory mechanism