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a The Samuel Roberts Noble Foundation, Inc., 2510 Sam Noble Parkway, Ardmore, Oklahoma, 73401
b USDA-ARS, 1680 Madison Avenue, Wooster, OH 44691
* Corresponding author (mian.3{at}osu.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCONCLUSIONSREFERENCES |
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Abbreviations: ABA, abscisic acid • AFP, antifreeze protein • AFLP, amplified fragment length polymorphisms • cM, centimorgan • EST, expressed sequence tag • kb, kilobase • MAS, marker-assisted selection • ORF, open reading frame • PAP, pokeweed antiviral proteins • PCR, polymerase chain reaction • PSII, photosystem II • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphisms • RMV, Ryegrass mosaic virus • QTL, quantitative trait loci • SNP, single nucleotide polymorphism • SSH, suppression subtractive hybridization • SSR, simple sequence repeat • STS, sequence-tagged sites
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCONCLUSIONSREFERENCES |
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In part because of the genetic complexity of forage and turf grasses, relatively little investment has been directed toward understanding and improving stress tolerance of these economically important species. Molecular technologies, such as genetic transformation and marker-assisted breeding, have become effective and efficient procedures for improving stress tolerance of some major crop species {e.g., soybean [Glycine max (L.) Merr.], rice, cotton [Gossypium hirsutum L.], and wheat}; however, conventional breeding through sexual hybridization is still the principal route for the development of stress-resistant forage and turf varieties. Several major factors contribute to the slow advancements in molecular and genomics research for improvement of stress tolerance in forage and turf grass species. First, most grasses are genetically complex, being out-crossing polyploids, and thus present highly complicated research targets. Second, most stress-resistance traits are under complex control of a number of genes and map as quantitative trait loci (QTL) throughout the genome (Fujimori et al., 2003; Yamada et al., 2004). Third, the lack of well-characterized genetic materials and absence of repeatable and efficient phenotyping protocols for many target traits limit the application of genomics technologies. Finally, the forage and turf grass community in general has failed to attract major funding for molecular and genomics research from either government agencies or private industries. Nevertheless, appreciable progress has been made in developing molecular and genomics tools for improvement of these grasses.
Biotic and Abiotic Stresses and Their Interactions
At one time or another during their life cycle, most plants encounter biotic and abiotic environmental stresses, which are two major factors that determine the distribution and productivity of crop plants. Thus, improvement of stress tolerance is a major plant breeding goal (Tolmay, 2001; Araus et al., 2002; Lecoq et al., 2004). Biotic stresses are caused by organisms such as bacteria, fungi, insects, nematodes, and viruses (Dangl and Jones, 2001). In contrast, abiotic stresses are caused by an unfavorable physical or chemical environment surrounding the plant. The mechanisms and physiology of abiotic stress tolerance of plants have been studied extensively (Levitt, 1972, 1980). Many factors interact to determine how plants respond to environmental stresses, including (i) the genetic constitution and developmental stages of the plants, (ii) the duration, frequency, and severity of the stress, and (iii) the additive or synergistic effects of multiple stresses (Bray et al., 2000). Failure to compensate for a severe stress may result in plant death. Plants respond to stress through different mechanisms, although most of them are not well understood (Bohnert et al., 1995). Information from model species, such as Arabidopsis and rice, whose genomes have recently been sequenced, and the use of microarray and other genomic technologies can provide insight into the complexities of the processes involved in plant stress tolerance (Moran et al., 2002; Shinozaki et al., 2003; Cooper et al., 2003; Xiong and Yang, 2003; Lian et al., 2004).
Molecular mechanisms involved in stress tolerance of forage and turf grasses are largely unknown, and studies in this area lag far behind other major crop species. The establishment of synteny among grass genomes based largely on colinearity of genetic markers aids the molecular analysis and manipulation of forage grasses (reviewed in Feuillet and Keller, 2002). Some evidence suggests that genes responsible for abiotic stress resistance in rice and other cereals have a similar role in forage and turf (Jiang and Huang, 2002; Humphreys et al., 2004). Numerous genomics research projects in forage and turf are underway, which should provide large amounts of functional information on the genes regulating agriculturally important traits (reviewed in Zhang and Mian, 2003).
In this review, we cover the recent molecular and genomics research aimed at improving stress tolerance of forage and turf. Most research work is currently being conducted in cool-season grasses, primarily ryegrass and fescue, but research on fungal resistance in bermudagrass, a warm season turf is also underway. We also discuss the economically important endophyte mediated stress resistance and bioprotection in forage and turf species. Stress tolerance and grass–endophyte interaction studies discussed in this review are summarized in Table 1.
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Commonly used molecular markers include restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs) or microsatellites, and more recently, single nucleotide polymorphisms (SNP). RFLP markers are highly reproducible codominant markers often conserved across plant species and genera. For this reason they have been used to study DNA conservation and synteny among related plant species. Even though RFLP markers are useful for dissecting plant genomes, their application in plant improvement is limited because the use of hybridization-based RFLP markers is slow and labor intensive and requires large amounts of DNA. The PCR-based markers overcome these limitations. The PCR-based RAPD markers are generated by using a single primer of arbitrary sequence. RAPDs are dominant markers; however, they are highly polymorphic, require very little DNA as a template, and can generate hundreds of markers at a low cost. The RAPD primers are commercially available at a relatively low price. The main limitation of RAPD markers is their reproducibility. The shortness of the primers requires that PCR amplifications are done under relatively low stringency conditions, thus increasing the chance of nonspecific priming or primer mismatch (Mueller and Wolfenbarger, 1999). The high error rate reduces reproducibility, making RAPDs unsuitable for some applications (Perez et al., 1998). For marker assisted selection, RAPD markers linked to a trait of interest need to be converted to more reproducible PCR-based markers, such as sequence characterized amplified regions (SCARs) (Paran and Michelmore, 1993), or sequence-tagged sites (STS) (Olson et al., 1989). Another marker based on primers of random sequence are AFLPs, which are relatively cheap, easy, fast, and reliable for generating hundreds of informative genetic markers, and unlike RAPD, they are highly reproducible (Vos et al., 1995; Mueller and Wolfenbarger, 1999). However, it is often necessary to convert AFLP markers into other PCR markers, for example, SCARs, for marker-assisted breeding (Paran and Michelmore, 1993; Xu et al., 2001b). The AFLPs are mostly population specific, and thus not useful for comparative mapping studies.
Simple sequence repeats or microsatellites are PCR-based codominant markers. Unlike RAPDs and AFLPs they are reproducible across populations within the same species and to some extent across species and genera. Currently, SSRs have the broadest application among all major marker systems available for plants. Simple sequence repeat marker analysis can be easily automated. The development of genomic SSR markers requires considerable investment of time and money in cloning and sequencing genomic DNA (Akkaya et al., 1992). However, many mRNA transcripts also contain SSRs, and the large-scale generation of DNA sequence from ESTs (see below) in many plant species results in the simple detection of EST-SSR markers (Saha et al., 2004). The SNPs (single nucleotide change and small insertion or deletions in DNA sequences) are by far the most abundant source of DNA polymorphism (Collins et al., 1998; Kwok et al., 1996). Cogan et al. (2005) have reported 16 SNPs kb–1 in the coding and 25 SNPs kb–1 in the noncoding regions of perennial ryegrass (Lolium perenne L.). High-throughput low-cost detection systems combined with the abundance of SNPs in plant genomes suggests that in the future SNPs will become the marker of choice for many plants species. Single nucleotide polymorphism markers developed from candidate genes could actually result in functional markers, that is, marker alleles that actually are the cause of phenotypic differences (Andersen and Lubberstedt, 2003).
Analyses of gene expression can be conducted by evaluating collections of ESTs developed from different plants and different tissues, or by expression profiling with DNA microarrays. ESTs are short (200–500 bp) DNA sequences generated from the 3' or 5' ends of random cDNA clones. Sequencing ESTs can be done rapidly, providing a "tag" for each functional gene in the genome. Expressed sequence tags can be mapped as genetic markers, used for comparative genomic studies through alignments of ESTs from related species, and used as a rich source of other molecular markers, for example, SSRs and SNPs. Expressed sequence tag data are commonly used to develop microarrays to analyze expression of thousands of genes simultaneously. Two platforms for gene expression analysis are currently available. One type, developed at Stanford University and commonly called DNA microarrays, uses a solid surface, such as glass, on which thousands of unique cDNAs are placed (or spotted) by a robot (DeRisi and Iyer, 1999). The other platform is an array of oligonucleotides (20- to 70-mer oligos) based on EST sequence data which are deposited on a solid support (or "chip") by either in situ synthesis or by conventional oligo synthesis followed by on-chip immobilization. This platform was developed and patented by Affymetrix (Santa Clara, CA) Although Affymetrix chips are expensive for most purposes, they are typically more sensitive than cDNA microarrays. Because sequencing of genomes, generation of ESTs, and production of microarrays are expensive, these technologies are not in widespread use in plant breeding programs at the current time.
RFLP markers have been developed from F. arundinacea Schreb. (Xu et al., 1991) and from Lolium (Hayward et al., 1998). The RFLP anchor probes developed from cereal grasses by van Deynze et al. (1998) can be used successfully in many forage and turf grass species. Simple sequence repeat and EST-SSR markers have been developed from tall fescue (Festuca arundinacea L.) (Saha et al., 2004, 2005a), ryegrasses (Jones et al., 2001, 2002a; Kubik et al., 1999), meadow fescue (Festuca pratensis Huds.) (Alm et al., 2003). Conserved grass EST-SSR markers developed from cereal grass species (Kantety et al., 2002) are also useful markers for forage and turf grass species (Saha et al., 2004, 2005b; Warnke et al., 2004). High density molecular maps composed of PCR-based markers have been constructed for ryegrasses (Jones et al., 2002a, 2002b; Warnke et al., 2004; Inoue et al., 2004), tall fescue (Saha et al., 2005c), and meadow fescue (Alm et al., 2003). More than 50 000 ESTs from ten cDNA libraries representing various tissue types and growth stages of tall fescue grown under various environmental stress factors have been developed by the Forage Improvement Division of the S.R. Noble Foundation (unpublished data, January 2005). We have used these ESTs for developing EST-SSR markers (Saha et al., 2004). These ESTs will also be used to develop SNP markers and microarrays. The Plant Biotechnology Center of Agriculture Victoria (La Trobe University, Melbourne, VIC) and their collaborators have been developing integrated resources for ryegrass functional genomics (Spangenberg et al., 2003). This group has developed more than 45 000 ESTs from perennial ryegrass representing approximately 15 000 unigenes that comprise a cDNA microarray. They also developed 10 000 ESTs and a cDNA microarray with 4000 unigenes from Neotyphodium grass endophytes. These ESTs are also being used for developing EST-SSR and SNP markers. Other SNP markers have also been developed for ryegrasses (Cogan et al., 2005; Humphreys et al., 2005b).
Defense Response to Biotic Stress
Biotic stresses can be caused by many organisms, including fungi, bacteria, viruses, and nematodes. On recognition of the attacking pathogens in some plants, defense systems are activated rapidly and each plant cell requires only minutes to switch from normal primary metabolism to a multitude of secondary metabolism defense pathways, activating novel defense genes and enzymes (Dievart and Clark, 2004). Plant defenses are usually associated with elicitor- and pathogen-mediated induction of gene expression. Cascades of transcription factors may be used to amplify the input signal or to modify the regulation of specific aspects of the complex plant defense response (Singh et al., 2002). Therefore, transcriptional activation of specific defense genes is important for the inducible resistance response. trans-acting DNA elements, often involved in the regulation of plant defense genes (Cooper et al., 2003), are regulated by either a rapid increase in transcript concentrations from steady state or changes in their phosphorylation status (Novillo et al., 2004). Some plant species have developed a special defense strategy through an incompatible reaction with pathogens that involves host cell death (hypersensitive response). The hypersensitive response is a rapid reaction, which usually occurs within 24 h of pathogen attack and leads either directly or indirectly to localized cell death, preventing further spread of the pathogen (Heath, 2000).
Biotic stress resistance is one of the most important goals in the improvement of forage and turf grasses due to the yield and/or quality losses these stresses cause (Fujimori et al., 2004). Further, the limited use of fungicides and insecticides in forage grasses—due both to high cost and concerns for livestock and human health and environmental pollution—means that the best approach for controlling diseases and pests in these grasses is the development of resistant cultivars (Rommens and Kishore, 2000).
Defense through Secondary Metabolites
Generally, the plant cell possesses both constitutive and inducible defense strategies toward pathogen infections. Many plants constitutively produce secondary metabolites with antimicrobial properties. These compounds are either present in their biologically active forms or are stored as inactive precursors that are converted to their active forms in response to pathogen attacks or mechanical injuries (Hammond-Kosack and Jones, 2000). One group of secondary metabolites, the phenylpropanoid compounds, is involved in both local and systemic signaling for induction of defense genes (reviewed in Dixon, 2004). Defensive functions are not restricted to a particular class of phenylpropanoid compound; the simple hydroxycinnamic acids and monolignols as well as the more complex flavonoids, isoflavonoids, and stilbenes are known to have such functions. The biosynthetic pathways of the major classes of phenylpropanoid compounds are now well established, and many of the corresponding genes have been cloned (Mansfield, 2000; Dixon, 2001). A biologically active triterpenoid saponin was identified in the roots of oat (Avena sativa L.), which is highly effective against Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici J. Walker, a serious pathogen for many cereal crops (Osbourn et al., 1994). A comparative analysis of the chromosomal regions containing major QTL in the phenylpropanoid–flavonoid pathway in maize, sorghum [Sorghum bicolor (L.) Moench], rice, wheat, and barley (Hordeum vulgare L.) indicated a general similarity among these Poaceae species in the organization of the pathway, with variation in the specific flavonoid products synthesized (McMullen et al., 1998).
Resistance Gene–Associated Defense Responses
Resistance (R) gene–mediated disease resistance in plants has been studied intensively. More than 30 R genes have been isolated from monocots (rice, maize, and barley) and dicots (Arabidopsis, potato [Solanum tuberosum L.], tobacco [Nicotiana tabacum L.], tomato [Lycopersicon esculentum Mill. var. esculentum], flax [Linum usitatissimum L.], pepper [Piper nigrum L.], lettuce (Lactuca sativa L.), and beet [Beta vulgaris L. ssp. vulgaris]) (Hulbert et al., 2001). Although these genes have resistance against a range of taxonomically unrelated pathogens, most of them encode members of the nucleotide-binding leucine-rich repeat class (Jones and Jones, 1997). Isolating R genes from the majority of forage and turf grasses has been complicated by their large, complex genomes, but only limited resources have been devoted for this purpose. However, the synteny of grass species genomes is potentially useful for R gene isolation. For example, the isolation of the nucleotide binding site family of resistance gene analogs was accomplished in Italian ryegrass (Ikeda, 2005). Using primers designed from sequence motifs conserved among R genes, 62 open reading frames (ORFs) were identified from 9344 PCR clones and defined as potentially functional resistance gene analogs from Italian ryegrass. Specific STS primer sets were designed from these ORFs and 50 sequences were used successfully to amplify fragments with expected size from Italian ryegrass as well as from tall fescue and meadow fescue (Ikeda, 2005).
Since the genome of Lolium is closely related to that of Festuca, R genes can potentially be introgressed from ryegrasses to meadow fescue and tall fescue and vice versa by hybridization. Although the hybrids between Lolium and Festuca species are usually male sterile with low female fertility (Jauhar, 1993), the androgenesis technique, which produces diploid offspring that carry nuclear chromosomes from only the male parent, was used to select and stabilize useful Lolium–Festuca gene combinations (Lesniewska et al., 2001; Thomas et al., 2003). Transfer of crown rust (caused by Puccinia coronata f. sp. lolii) resistance from meadow fescue and tall fescue to Italian ryegrass using amphidiploid cultivars has been reported. The intergeneric hybrids were highly resistant to the pathogen (Oertel and Matzk, 1999).
QTL for Disease Resistance
Identification of molecular markers linked to QTL for disease resistance could greatly improve the potential to develop cultivars resistant to complex diseases. Curley et al. (2004) identified QTL for gray leaf spot [caused by Magnaporthe grisea (T.T. Herbert) Yaegashi & Udagawa] resistance in two genomic regions of perennial ryegrass, and one of the regions appears syntenic with rice linkage group 7. Other examples include identification of QTL for crown rust resistance in perennial ryegrass (Barre et al., 2000; Roderick et al., 2002; Dumsday et al., 2003) and Italian ryegrass (Fujimori et al., 2003). A major QTL for crown rust resistance was mapped on linkage group 2 in perennial ryegrass using a two-way pseudo-testcross mapping population developed by crossing two ryegrass parents with intermediate resistance against a pathogen population (Dumsday et al., 2003). Fujimori et al. (2003) identified 34 AFLP markers covering a 36-cM region flanking the resistance gene locus Pc1 in Italian ryegrass. A resistance gene analog marker cosegregating with Pc1 was developed (Fujimori et al., 2003).
Functional Genomics and Genetic Transformation
Genomic approaches have been used to investigate gene functions in forage and turf species. For example, chitinase function was characterized on suppressing summer patch disease (caused by Magnaporthe poae Lanschoot and Jackson) in Kentucky bluegrass with a mutant strain C5 of Stenotrophomonas maltophilia (Kobayashi et al., 2002). cDNA-derived macro- and microarrays were used to identify disease-resistance genes and to confirm their expression. In another study, suppression subtraction hybridization (SSH), sequencing of cDNA clones from forward and reverse normalized libraries, and cDNA microarrays were used to identify genes associated with resistance or susceptibility to a fungal disease, spring dead spot, caused by Ophiosphaerella herpotricha (Fr.) Walker in bermudagrass (Guenzi and Zhang, 2004). After fungal infection, genes involved in signaling pathways and the oxidative burst defense mechanism, including ethylene receptor, rac GTPase activating protein, DnaJ protein, voltage-dependent anion channel protein, eukaryotic translation initiation factor, ADP-ribosylation factor–like protein, and LLS1 protein, had significantly higher expression levels in resistant than susceptible genotypes (Guenzi and Zhang, 2004). The first forage grass gene chip was constructed recently by Agriculture Victoria and AgResearch, New Zealand (Spangenberg et al., 2004). This cDNA-based microarray represents 14 767 unigenes generated from 28 cDNA libraries from ryegrass, and will be useful in novel gene expression profiling and promoter discovery in ryegrass and closely related species, for example, tall fescue.
Disease-resistance genes that control plant defense networks can be transferred to forage and turf species to improve resistance (reviewed in Wang et al., 2001). A large number of independently transformed lines that show stable transgene integration and expression must be produced for practical use in breeding program. A good transformation system is required for the rapid production of large numbers of independently transformed lines with high disease resistance. Takahashi et al. (2002) have recently developed a transformation technique based on the particle gun method using the Italian ryegrass cv. Waseaoba, which is susceptible to crown rust, indicating that this system may be useful for confirming the function of the R gene in Italian ryegrass. Transgenic creeping bentgrass plants expressing three forms of ribosome-inactivating pokeweed antiviral proteins (PAP) were generated to increase the resistance to dollar spot disease caused by the fungal pathogen Sclerotinia homoeocarpa F.T. Bennett (Dai et al., 2003). Some PAP transformants could accumulate stable levels of the protein, but had symptoms of toxicity; one low-expressing line with normal phenotype exhibited good disease resistance. RNA-mediated virus resistance has been used to improve the disease resistance of forage species. The resistance of perennial ryegrass to Ryegrass mosaic virus (RMV) has been improved by transformation with an RMV coat protein gene (RgMV-CP) using particle bombardment (Xu et al., 2001a). The genetic background of transgenic line, virus strain, and the period after inoculation influenced the RgMV level. Molecular analysis indicated that RgMV resistance operates by targeted RNA degradation, resulting in post-transcriptional gene silencing along with inhibition of virus RNA replication.
Tolerance and Adaptation to Abiotic Stress
Under natural condition, plants are often subjected to several environmental stresses synchronously (e.g., heat and drought), although damage is usually caused by the most severe factor. However, many stresses induce a plant response that results in similar physiological changes. For example, drought stress, salt stress, and cold stress all involve problems of water availability (Seki et al., 2002). Changes in the redox state of photosystem II (PSII) are related to extreme temperatures, including freezing and heat (Adams et al., 2002; Zhang et al., 2005). Therefore, exposure to one type of stress may induce a degree of tolerance to other stresses because of such overlaps.
Stress conditions may cause instability of the genome. In response to temperature changes, the allohexaploid F. arundinacea genome changed the numbers of interspersed DNA repeats when seedlings were exposed to 10 or 30°C (Ceccarelli et al., 2002). The relevant sequences were dispersed along the length of all chromosomes, but concentrations around the centromeric regions were evident on certain chromosomes. Genome plasticity could be an important mechanism that allows species like F. arundinacea to grow in a wide range of environmental conditions and climates (Ceccarelli et al., 2002).
Heat Stress
High temperature and drought are two of the major abiotic stress factors that affect plant distributions (Guy, 1999). Plants are often under high-temperature stress even in their native habitats. Higher plants exposed to excess heat exhibit a characteristic set of cellular and metabolic responses, including a decrease in the synthesis of normal proteins and an accelerated transcription and translation of heat shock proteins (Morimoto et al., 1994). High temperature may also cause large, reversible effects on the rate of photosynthesis (Weis and Berry, 1988). In addition to altering patterns of gene expression, heat also damages cellular structures, including organelles and the cytoskeleton, and impairs membrane function. High soil temperature is shown to be one of the limiting factors for forage production and turf management of many cool-season forage and turf species grown in temperate–tropical zones (Wallner et al., 1982).
Plants can acquire thermo-tolerance if subjected to a nonlethal high temperature for a few hours before encountering heat-shock conditions. The increase in thermo-tolerance above the basal level is caused by acclimation (Vierling, 1991). An acclimated plant with cellular and metabolic changes can survive exposure to a temperature that would otherwise be lethal (Hong et al., 2003). Plants surviving heat-stress conditions have developed this thermo-tolerance mechanism, a major factor governing the distribution of plants around the globe (Woodward, 1988). Acclimation to nonlethal heat treatment, therefore, may alter transcriptional regulation in a manner different than that caused by heat shock (Guy, 1999).
Recently, the molecular basis of heat tolerance and acclimation in Festuca sp. was studied in our group (Zhang et al., 2005). Heat tolerance-related gene transcripts were cloned using SSH between a heat-tolerant and a heat-sensitive fescue genotype exposed to high temperatures (up to 44°C). Three subtractions were conducted between samples of the two genotypes collected after 12 h of exposure to 39, 42, and 44°C. Under heat stress conditions, cell maintenance and genes related to chloroplasts, photosynthesis, defense, protein synthesis, signaling, and transcription factors had higher expression levels in the heat-tolerant genotype. Genes related to metabolism and stress had higher expression in the heat-sensitive genotype. These results indicate that a balance between energy generation (photosynthesis) and metabolism (photorespiration) may be involved in heat tolerance of fescue plants (Zhang et al., 2005).
Stresses Involving Water Deficit
Water deficit stresses can affect plant growth significantly if the quantity or quality of water available is insufficient to meet basic needs. Besides drought, salinity and low or high temperatures can also result in water stress. Many factors can affect the response of a plant to water-deficit stresses, including the duration of water deficit, the rate of onset, and the acclimation to water stress by previous exposure. Water potential, relative water content, and transpiration rate are important parameters describing the water status of a plant. Expression analysis of drought, cold, and salt-inducible genes has shown there might be some regulatory systems that are in common among these stresses. Using cDNA microarrays, Seki et al. (2002) monitored the expression profiles of 7000 Arabidopsis genes under drought, cold, and high-salinity stress and found in total 227 drought-inducible, 53 cold-inducible, and 194 high salinity– inducible genes. Among these genes, 22 were induced by all three stresses (Seki et al., 2002). Thirty genes were induced by cold and drought stress, and 24 by cold and salt-stress. However, 70% of the salt-induced genes were also induced by drought stress, indicating strong relationships between plants' responses to these two stress factors.
Drought Stress
Transient or prolonged drought conditions reduce the amount of water available for plant growth. In response to drought stress, physiological and biochemical changes may result in alteration of protein synthesis or degradation (Riccardi et al., 1998). The accumulation of drought-induced proteins and physiological adaptations to water deficit may influence drought tolerance (Han and Kermode, 1996; Riccardi et al., 1998). Improved cultivars of forage for arid lands have drought-resistance mechanisms that enable them to survive and grow in areas with low moisture availability. Traditional breeding has generated improved drought-resistant forage species by combining genes from closely related species, for example, the allotetraploid hybrid between L. multiflorum and F. glaucescens Hegetschw. & Heer, one of the ancestral species of F. arundinacea, was developed in France as a potential cultivar (Ghesquière et al., 1996), which combines good agronomic characters of Lolium with the drought resistance of Festuca. Recently, Humphreys et al. (2005a) reported introgressing drought resistance genes from F. glaucescens (2n = 4x = 28) into L. multiflorum (2n = 2x = 14). A diploid Lolium genotype with a single F. glaucescens introgression event located distally on the nucleolar organizer region arm of chromosome 3 was produced. Amplified fragment length polymorphisms and STS markers were used to monitor the intogression of F. glaucescens (Humphreys et al., 2005a).
The dehydrin family of proteins, which range in size from 9 to 200 kDa, can accumulate in a wide range of plant species under dehydration stress (Close, 1996). Dehydrins are hydrophilic and heat stable and may protect other proteins and help maintain the physiological integrity of cells (Bray, 1993). Drought-induced protein synthesis and drought-regulated dehydrin gene expressions were observed by Jiang and Huang (2002) in two tall fescue cultivars, Southeast and Rebel Jr. The effects of abscisic acid (ABA) application on the drought tolerance of the cultivars were also evaluated. Dehydrins accumulated in both drought-stressed and ABA-treated plants of both cultivars, which could protect plants from further dehydration damage. The protective effects of ABA on the enhancement of physiological activities under drought stress paralleled the delayed induction of protein synthesis in tall fescue. However, the two tall fescue cultivars did not differ in their responses to ABA. Therefore, increased drought tolerance resulting from ABA application might not be directly related to the accumulation of a higher level of dehydrins or to the production of a unique dehydrin protein (Jiang and Huang, 2002).
Freezing Stress and Cold Acclimation
Subzero temperatures can cause a type of water-deficit stress by affecting cellular water relations. As ice formation is initiated in the intercellular spaces, cellular water starts moving toward extracellular space due to the water potential gradient. Therefore, cellular dehydration develops within the cell in response to freezing. Plant survival in low temperature during winter is genotype specific and known as winterhardiness. Winterhardiness is a complex trait (Guy, 1990) and requires the development of cold acclimation by exposing plants to low nonfreezing temperatures, typically 0 to 10°C, and shortened photoperiod (Thomashow, 1999). A gene expression analysis during cold acclimation in bermudagrass indicated the induction of chitinases in response to both low temperature and dehydration stresses (de los Reyes et al., 2001).
The mechanisms of cold acclimation are not well understood. However, several processes that occur during acclimation include slowed or arrested growth; reduced tissue water content; altered cell pH; protoplasm viscosity and photosynthetic pigments; reduced ATP levels (Levitt, 1980); transient increases in ABA (Chen et al., 1983); changes in membrane lipids (Uemura and Steponkus, 1994); accumulation of compatible solutes including proline, betaine, polyols and soluble sugars; and accumulation of antioxidants (Tao et al., 1998). Eagles et al. (1993) reported that temperate grasses store fructans, a soluble polymer capable of rapid polymerization and depolymerization, which can help the recovery of freezing in Avena sativa, Lolium perenne, and L. multiflorum Lam. The partitioning of solutes may be important as survival depends on survival of apices, particularly the lateral buds, rather than mature leaf tissue (Eagles et al., 1993).
Some freezing-tolerant grass species, such as rye (Griffith et al., 1992), accumulate antifreeze proteins in the apoplast to slow the formation of ice and retard cellular dehydration (Marentes et al., 1993). A heat-stable antifreeze protein (AFP) has been isolated from L. perenne (Sidebottom et al., 2000). This protein, which contains a seven-residue repeating motif, causes only slight thermal hysteresis (temperature gap between the melting and freezing points of ice was only 0.1°C) but is very efficient at inhibiting ice recrystallization. Kuiper et al. (2001) have constructed a three-dimensional (3-D) model of the L. perenne (Lp) AFP based on the structures of fish and insect AFPs and the primary sequence of the protein. In this study two ice-binding sites were predicted on either side of the AFP, with complementarity to the prism face of ice. The superior ice recrystallization inhibition activity of LpAFP may be due to unusual duplication of putative ice-binding sites on opposite sides of the protein (Kuiper et al., 2001). Recently, a homeologous F. pratensis antifreeze protein sequence has been isolated from a bacterial artificial chromosome library; the sequence of this protein is encoded by two closely linked loci on F. pratensis chromosome 2 (Humphreys et al., 2004).
Festuca pratensis carries a number of valuable traits for adaptation to harsh winter conditions that can be introgressed into Lolium to improve its winter hardiness (Humphreys et al., 1998). Canter (2000) reported that ice tolerance had been introduced from F. pratensis to L. perenne. Freezing tolerance of L. perenne was enhanced by an introgression from chromosome 3 of F. pratensis onto a homeologous Lolium chromosome (Humphreys et al., 2004). In addition, plants with consistently good winter hardiness and freezing-tolerance were found in introgression lines derived from other L. perenne x F. pratensis crosses (Humphreys et al., 2004).
Changes in the redox state of PSII have been proposed as a temperature-sensing mechanism for cold acclimation (NDong et al., 2001; Rapacz, 2002). Low temperatures may cause photoinhibition if the rate of light harvesting by PSII exceeds the capacity of electron transport. Therefore, cold-tolerant plants can maintain photosynthesis and reduce the potential for photoinhibition by dissipating excess energy harvested by PSII (Huner et al., 1998; Adams et al., 2002). Photosynthetic acclimation and resistance to high light–induced inactivation of PSII at low temperature were studied on androgenic plants generated from a Festuca pratensis x Lolium multiflorum (4x) cross using chlorophyll fluorescence techniques (Rapacz et al., 2004). Increased energy dissipation before winter through a lower maximum quantum yield of PSII correlated with improved winter survival of these plants. Winter hardy plants were more resistant to cold-induced inactivation of PSII in controlled conditions (Rapacz et al., 2004). Representational difference analysis was used to selectively amplify cDNA fragments from cold-induced F. pratensis seedlings and to identify cDNAs upregulated during cold acclimation (Canter, 2000). Homologs of small ribonucleic proteins and the chloroplast encoded gene psbA which codes for the D1 protein of PSII were found to be induced by low temperatures.
Transformation of freezing tolerance–related genes were used to produce forage and turf with high freezing tolerance. A fructosyltransferase (LpFT1) cDNA and its promoter region was isolated from perennial ryegrass (Chalmers et al., 2003). Transgenic perennial ryegrass overexpressing wheat fructosyl transferase genes, wft1 and wft2 under CaMV 35S promoter, showed increased tolerance to freezing on a cellular level and accumulated greater amount of fructan than nontransgenic plants (Hisano et al., 2004). Therefore, manipulation of FT gene expression enabled its functional analysis in planta and may be a useful strategy to produce water stress–tolerant grasses. Another cold tolerance–related gene, ipt from Agrobacterium tumefaciens that codes for isopentenyl transferase was transformed into turf-type tall fescue (Hu et al., 2005). The transgenic turfgrass had increased tillering ability and cold tolerance. The transgenic plants remained more vigorous and stayed green longer under lower temperatures compared to control plants (Hu et al., 2005).
A number of QTL related to freezing tolerance and winter survival have been identified. Two major QTL for freezing tolerance and four QTL for winter survival were identified for F. pratensis (Rognli et al., 2002). Heterologous wheat anchor probes indicated that Frf4_1 on LG4 of F. pratensis was orthologous to the frost-tolerance loci Fr1 and Fr2 in wheat. Genetic linkage mapping has identified the same markers linked to QTL for multiple stresses, suggesting the existence of clusters of genes, or same genes controlling different stress-tolerance processes, especially for the stresses that have common mechanisms such as frost and drought. For example, QTL for tolerance to several stress condition were found on chromosomes 1, 2, 5, and 6 in meadow fescue (Rognli et al., 2002). In the same study, comparative mapping with Triticeae species (wheat, barley, and rye), Lolium, oat, rice, maize, and sorghum have showed that the QTL on chromosome 1 for winter survival was orthologous to a water-soluble carbohydrate QTL in Lolium. A putative QTL for resistance to moderate drought was found on chromosome 6 and may be orthologous to a mapped ABA locus in wheat. This study also reported several genes on chromosome 3 for survival against severe drought (Rognli et al., 2002). Guthridge et al. (2003) conducted a molecular marker–based analysis of drought-associated traits in perennial ryegrass. QTL for the correlated morphological traits (root and shoot length) and regulation of water soluble carbohydrate (e.g., fructan) accumulation have been assigned to linkage group 2. Recently, Yamada et al. (2004) reported QTL analysis of perennial ryegrass for several economically important traits, including winter hardiness. Although there were no significant QTL identified for winter survival in the field, a QTL for electrical conductivity corresponding to frost tolerance was located close to the flowering time locus on LG 4 of L. perenne.
Endophyte-Associated Resistance
Epichloë–Neotyphodium endophytes are a group of biotrophic fungi that form symbiotic associations with temperate grasses of the subfamily Pooideae (Schardl and Wilkinson, 2000). These endophytes systemically colonize the intercellular spaces of leaf primordia, leaf sheaths, and leaf blades of vegetative tillers and the inflorescence tissues of reproductive tillers (Christensen et al., 2002). In general, endophyte-infected (E+) grass genotypes outcompete noninfected (E–) genotypes, and are also strong competitors to other plant species under diverse biotic and abiotic stresses (reviewed in Malinowski and Belesky, 2000; Schardl et al., 2004). The ability of Epichloë–Neotyphodium endophytes to synthesize a range of secondary metabolites in planta constitutes a major ecological benefit for the symbiotum (Schardl, 1996). Metabolites identified to date include both anti-insect (peramine and lolines) and antimammalian (ergot alkaloids and indole–diterpenes) compounds (Bush et al., 1997).
Endophyte-infected grasses are more competitive than noninfected grasses under low-input environments (Hill et al., 1998). Kelemu et al. (2001) isolated an endophytic fungus, Acremonium strictum/kiliense W. Gams from a commercially important forage of tropical America, signalgrass [Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster]. The endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants after both were artificially inoculated with the pathogenic Drechslera fungus. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte (Kelemu et al., 2001). Endophyte-infected tall fescue plants had increased resistance to Russian wheat aphid [Diuraphis noxia Mord.] (Clement et al., 1996) and bird cherry-oat aphid (Rhopalosiphum padi L.) (Clement et al., 2001; Bultman et al., 2004). Therefore, presence of antifungal or anti-invertebrates compounds may be a common feature of biotic responses of these endophyte–grass associations.
In some cases, endophyte-infected grasses may have direct chemical effects on the competitor. Neotyphodium lolii (Latch, Christensen, and Samuels) Glenn, Bacon, and Hanlin produces alkaloid compounds that effectively protect perennial ryegrass from the Argentine stem weevil (Listronotus bonariensis Kuschel) in New Zealand (Prestidge and Gallagher, 1988). The endophyte alkaloid that is primarily responsible for feeding deterrence was peramine (Rowan and Latch, 1994). This compound was also correlated with activity against the aphid Schizapus graminis (Siegel et al., 1990). Another group of anti-insect alkaloids, known as loline, are saturated 1-aminopyrrolizidine alkaloids. They have broader range and more overt toxicity to insects than peramine (Bush et al., 1993). Alkaloids are always present in resistant plants, suggesting the corresponding genes are constitutively expressed. Nevertheless, there is also evidence that endophytes may mediate an induced host response since loline alkaloid levels increased dramatically in response to clipping (mock herbivory) (Craven et al., 2001). Although the endophyte is barely present in roots (Azevedo and Welty, 1995), Popay et al. (2004) found that a new endophyte isolate suppressed root aphid, Aploneura lentisci Pass., in perennial ryegrass. Endophyte-infected plants also showed defense responses to nematode attack. For example, Neotyphodium coenophialum (Morgan Jone and W. Gams) Glenn, Bacon, and Hanlin dramatically reduced root-knot nematodes (Meloidogyne marylandi Jepson and Golden) and the migratory nematode Pratylenchus scribneri Steiner (Elmi et al., 2000; Kimmons et al., 1990) in tall fescue.
The competitive advantage of endophyte-infected grasses over the uninfected grass in adverse environmental conditions such as drought and heat are well known, although the mechanisms are not understood. It may result from morphological responses such as development of more numerous tillers (Hill et al., 1990), greater leaf elongation rate (Malinowski et al., 1997), and altered root architecture (Malinowski et al., 1999). Medvescigh et al. (2004) reported that endophyte-infected plants were taller with longer leaves and with fewer but thicker tillers under water stress compared to endophyte-free plants. The effects of endophytes on stress tolerance, including osmotic adjustment (Belesky et al., 1989), water relations (Cheplick et al., 2000), and drought recovery (Morse et al., 2002) have been reported. Endophyte-infected tissues accumulate drought-protective osmolytes (Richardson et al., 1992) and activate stress-related proteins including dehydrin (Carson et al., 2004), POD (peroxidase), SOD (superoxide dismutase), and PPO (polyphenol oxidase) (Chen et al., 2001). Under water stress, the tall fescue endophyte was also associated with a significant increase in cell wall elasticity (White et al., 1992). Neotyphodium-infected perennial ryegrass showed less severe reduction in relative water content compared to endophyte free plants (Hahn et al., 2004). However, Northern blot analysis and RT-PCR indicated lolitrem B and ergovaline biosynthesis genes, ltmG and lpsA, did not alter significantly in response to water deficit, which suggested the mechanism responsible for increased water deficit tolerance did not involve changes in known alkaloid levels (Hahn et al., 2004).
In E+ ryegrass and tall fescue, alkaloid concentration in planta was influenced by plant genotype, tissue type, season, and various abiotic environmental conditions and associated biotic responses (Ball et al., 1995; Easton et al., 2002; Hiatt and Hill, 1997). Plant genotype plays an integral role in determining the outcome of the host plant x endophyte x insect interactions, primarily by modifying the expression of alkaloids produced (Easton et al., 2002). Seasonal changes in endophyte concentration can reduce the effectiveness of the endophyte-mediated resistance to Argentine stem weevil (Popay and Mainland, 1991). Temperature and seasonal factors also interact with the endophyte to alter the level of resistance in E+ ryegrasss to the greenbug [S. graminum (Rodani)] (Breen, 1992). Therefore, combining the right fungal isolate with a complementary host genotype could target bioactivity and optimize plant performance of turf and forage grasses (Popay et al., 2004).
Indole-diterpene and ergot are the other two classes of endophyte alkaloids. They possess both anti-insect and antivertebrate activities and are the causes of problems in livestock such as ryegrass staggers and fescue toxicosis (Lacey, 1991). Recent approaches to endophyte x grass interactions focus on isolated endophyte strains that show minimal or no production of alkaloids toxic to livestock yet retain the pest- and drought-resistance benefits of symbiotic plants (Parish et al., 2003a, 2003b). These associations are crucial for improved livestock performance on forage species (Schardl et al., 2004).
Because of the biological importance of endophyte secondary metabolites, genes encoding endophyte indole-diterpenes (loline, ergot alkaloid, paxilline, and lolitrem) biosynthesis have been identified (reviewed in Scott, 2004). Studies on gene expression changes in response to biotic and/or abiotic stresses, and to identify the molecular basis of signaling pathways that occur between endophytes and hosts, are in progress (Spiering et al., 2003; Scott, 2004; Felitti et al., 2004). Cloning of these endophytic secondary metabolite genes will improve our understanding of the interaction between endophytes and their grass hosts involved in plant bioprotection mechanisms and enabling screening of field isolates for the presence, distribution and expression of these genes (Young et al., 2001; Lodwig et al., 2003). A knockout mutant of Neotyphodium was developed recently in which the ergot alkaloid biosynthesis gene dmaW was eliminated (Machado et al., 2004). Molecular manipulation of endophyte genes may produce isolates that are desirable for use in pastures to overcome animal toxicosis problems associated with endophyte toxins, yet retain their bioprotective features like the ability to resist insect herbivory (Fletcher, 1999; Scott, 2004).
Recently, the Molecular Plant Breeding Cooperative Research Center (MPBCRC) in Australia developed the world's first grass endophyte unigene array—the EndoChip, for the use of high throughput microarray analysis (Spangenberg et al., 2004). This endophyte microarray contains 20K unigenes of N. lolii and N. coenophialum origins. It is a tool for comparative gene expression studies between endophyte species, as well as a tool for high-throughput parallel gene expression profiling in grass endophytes.
Expressed sequence tag data for the grass fungal endophyte species N. lolii and N. coenophialum has been used to generate a set of SSR markers for the genetic analysis of the grass-endophyte symbiosis. Fifty SSR primer pairs from N. coenophialum and 57 from N. lolii were designed and evaluated using 20 Neotyphodium and Epichloë isolates. Analysis of SSR polymorphism identified 188 different alleles from 34 N. coenophialum derived EST-SSR loci and 280 different alleles from 45 N. lolii derived EST-SSR loci across the 20 endophyte isolates, although the polymorphism levels were low within N. coenophialum and N. lolii (van Zijll de Jong et al., 2003). Further study with the same group of endophytes assembled 22 EST-SSR markers into multiplex panels for in planta detection and genetic polymorphism analysis of the endophytes (Forster et al., 2004). The markers were shown to be endophyte specific and effective at low mass ratios of endophyte to plant genomic DNA. They could be used to investigate divergence between different endophyte species.
Interactions among Plant Stress Responses
Since a plant in the field is generally exposed to multiple stress factors simultaneously, it is important to consider the contribution of each factor to a resistance or defense response with all potential interactions, synergistic and antagonistic (Chinnusamy et al., 2004). Many studies have revealed interactions between pathogen responses and abiotic stress resistances (Reymond et al., 2000; Cheong et al., 2002; Roberts et al., 2002; Singh et al., 2002; Hegedus et al., 2003; Ludwig et al., 2004).
Two examples in the forage and turf literature highlight these interactions. First, the symptoms of bermudagrass spring dead spots increased under lower temperature (Guenzi and Zhang, 2004). Gatschet et al. (1996) demonstrated increased levels of chitinase, which is well known for its role as pathogenesis-related protein in the crowns of freeze-tolerant bermudagrass. Three bermudagrass chitinase genes known to be induced by cold acclimation and dehydration stresses (drought and ABA) were cloned (de los Reyes et al., 2001). One of these bermudagrass chitinase genes homologs (chitinase I) was also induced by fungal infection and showed more than twofold increase in expression when examined on a bermudagrass cDNA array (Zhang, 2002). This suggests that a pathogen attack is a combination of both physical stress and elicitor production (Farmer, 2000). A second example of dual role defense responses is O-methylation of phenolic compounds. NDong et al. (2003) reported the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of daphnetin in winter rye (Secale cereale L., Gramineae, cv. Musketeer). Although the importance of this enzyme in disease defense is well known (Dixon, 2001), transcript and corresponding enzyme activity were up-regulated by both low temperature and PSII excitation pressure (NDong et al., 2003). Therefore, access to a large body of information on the behavior of defense genes in multiple biotic or abiotic conditions might help in deciphering the intricate network of signaling pathways that link pathogens to specific responses of forage and turf.
There is considerable interest in defining the different transcription factors and phytohormones involved in the regulation of defense response pathways. Several reports have described the types of transcription factors that regulate the expression of genes under stress conditions in plants (Singh et al., 2002; Cooper et al., 2003; Novillo et al., 2004). It is likely that some of these factors are also involved in the transcriptional control of the same pathways during plant development (Cooper et al., 2003). Overexpression of stress-inducible transcription factor DREB1A in bahiagrass (Paspalum notatum Flüggé) showed enhanced freezing and drought tolerance (James et al., 2004). Exogenous application of ABA to several plant species including tall fescue induced a number of dehydrin-like proteins during dehydration or when under drought stress (Pelah et al., 1997; Jiang and Huang, 2002).
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Received for publication September 27, 2004.
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akinskiene, A.R. James, and H. Thomas. 1998. Physically mapping quantitative traits for stress-resistance in the forage grasses. J. Exp. Bot. 49:1611–1618.
wierzykowski, A. James, H. Thomas, and M.W. Humphreys. 2001. Androgenesis from Festuca pratensis x Lolium multiflorum amphidiploid cultivars in order to select and stabilize rare gene combinations for grass breeding. Heredity 86:167–176.[Web of Science][Medline]This article has been cited by other articles:
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  C. J. Nelson and J. C. Burns Fifty Years of Grassland Science Leading to Change Crop Sci., September 8, 2006; 46(5): 2204 - 2217. [Abstract] [Full Text] [PDF]
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