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Identification of molecular markers linked to physiological traits for stress tolerance is important for understanding the underlying genetics and breeding for improving plant stress tolerance. The objectives of the current study were to evaluate genotypic variations in a population of creeping bentgrass (Agrostis stoloniferaL.) germplasm for summer performance and drought tolerance under field conditions, and to validate candidate gene and quantitative trait locus (QTL) markers associated with physiological traits for stress tolerance in two locations differing in climate conditions. The trials were conducted in 2014 and 2015 in field plots covered under automatic rainout shelters in Georgia and New Jersey. Phenotypic and physiological traits, including visual turf quality ratings, membrane stability, chlorophyll content, normalized difference vegetative index, and leaf water content, were evaluated for heat tolerance during summer months and for drought tolerance. Using association analysis, phenotypic and physiological traits were measured to confirm linkage to 29 simple sequence repeat (SSR) markers and five candidate gene markers. Significant associations between traits and markers were found for 24 of the 34 molecular markers for the bentgrass population exposed to summer heat stress or drought stress. Those markers with significant associations may be useful in marker‐assisted selection and understanding how specific genetic regions influence abiotic stress tolerance in creeping bentgrass.

Identification of quantitative trait loci (QTL) for drought tolerance will allow for detection of important genomic regions associated with specific drought tolerance traits. This study aimed to identify QTL for drought tolerance traits in a colonial bentgrass (Agrostis capillarisL.) × creeping bentgrass (Agrostis stoloniferaL.) hybrid backcross population. The traits included relative water content (RWC), turf quality (TQ), electrolyte leakage (EL), chlorophyll content (CHL), normalized difference vegetation index (NDVI), and canopy temperature depression (CTD). A colonial × creeping bentgrass hybrid backcross population segregating for drought tolerance was exposed to drought stress by withholding irrigation in three different locations or environmental conditions (two greenhouse studies in two different years, 2009 and 2010, and a growth chamber study in 2010). Phenotypic trait data were collected in these studies. Significant variation in the parents and the progenies occurred for phenotypic traits and the broad‐sense heritability values indicated that the traits were polygenic in nature. A total of 32 potential QTL of varying effects (31 major and 1 minor) were detected on seven chromosomes. Significant overlap of QTL was found, particularly for CHL and CTD on 1A1, CTD, CHL, EL, and NDVI on linkage group 2A1, CTD and TQ on 2A2, TQ and RWC on 5A1 and 5A2, and TQ and CHL on 5A2. The QTL with the strongest effect for each trait (highest likelihood of the odds [LOD] and percent of phenotypic variance explained by the QTL) were CTD (4.19, 18.8%), CHL (4.23, 18.9%), EL (3.25, 14.9%), and NDVI (4.12, 22.4%) on group 2A1, RWC (3.27, 15.7%) on group 5A1, and TQ (4.38, 19.5%) on group 5A2. The analysis of potential genes in the QTL regions for these physiological traits indicate that the QTL may be linked to metabolic factors involved in N metabolism and energy metabolism such as photosynthesis and respiration. The QTL regions identified here could contain important genetic factors conferring drought tolerance in bentgrass species.

Transgenic herbicide‐resistant cultivars of creeping bentgrass (Agrostis stoloniferaL.) have been developed and are being considered for commercial release. Concerns regarding transgenic species include potential for outcrossing of the crop, which may produce novel genotypes with increased weediness. Persistence and multiyear growth of interspecific hybrids between creeping bentgrass and velvet bentgrass (A. caninaL.), colonial bentgrass (A. capillarisL.), dryland bentgrass (A. castellanaBoiss. and Reut.), and redtop bentgrass (A. giganteaRoth) and the backcross progeny of the hybrids were compared with the parental species in field experiments. The plants were evaluated over 2 yr in residential Kentucky bluegrass (Poa pratensisL.) and a low maintenance roadside turf of mixed species. With the exception of redtop bentgrass by creeping bentgrass interspecific hybrids, all interspecific hybrids between creeping bentgrass and colonial, dryland, and velvet bentgrasses were equally or less competitive relative to their parental species. Backcross progeny of interspecific hybrids, including those of redtop bentgrass by creeping bentgrass hybrids, were equally or less competitive than parental species. Bentgrasses that were highly competitive in residential turf were also competitive under roadside maintenance, suggesting bentgrasses have weediness potential regardless of management intensity.

Interspecific hybridization is a widely used approach to improve crop species. We are pursuing the possibility of using interspecific hybridization between the turfgrass species colonial bentgrass (Agrostis capillarisL.) and creeping bentgrass (A. stoloniferaL.) for the improvement of resistance to the fungal disease dollar spot (Sclerotinia homoeocarpaF.T. Bennett) in creeping bentgrass. From a field evaluation of a backcross population, it appears that introgression of dollar spot resistance from colonial bentgrass to creeping bentgrass is possible. We used the backcross population to generate the first genetic linkage map for colonial bentgrass. The map length was 1156 cM and consisted of 212 amplified fragment length polymorphic markers and 110 gene‐based markers. Colonial bentgrass is an allotetraploid species (2n= 4x= 28, A1and A2subgenomes). The map consisted of the expected 14 linkage groups, which could be assigned to either the A1or A2homoeologous subgenomes. Although there were dollar spot resistant individuals in the mapping population, no quantitative trait loci associated with resistance were detected.

Many wild and cultivated cool-season grass species are naturally infected with fungal endophytes of the genera Neotyphodiumand Epichloë.These associations generally are considered mutualistic with the plants benefiting from reduced herbivory and the fungi benefiting from nutrients supplied by the plants. The fungi secrete proteins that might have a role in the interspecies symbiosis. In the interaction between Poa amplaMerr. and the endophyte Neotyphodiumsp., a fungal chitinase was detected in the apoplastic protein fraction. The chitinase was also the major protein secreted in culture. Sequence analysis of the chitinase revealed it has a low level of amino acid sequence identity to other fungal chitinases and one of the conserved active site residues is altered. DNA gel-blot analysis indicated the chitinase was encoded by a single gene. Expression of similar chitinases also was detected in endophyte-infected tall fescue (Festuca arundinaceaSchreb.), perennial ryegrass (Lolium perenneL.) and Chewings fescue (Festuca rubraL. subsp. fallax[Thuill] Nyman). This is the first report of an endophyte chitinase expressed in the infected host grass. As a secreted hydrolytic enzyme, the chitinase might have roles in the nutrition, growth or defense of the endophyte.

Mutualistic fungal endophytes infect many grass species and often confer benefits to the hosts such as reduced herbivory by insects and animals. The physiological interactions between the endophytes and their hosts have not been well characterized. Fungal-secreted proteins are likely to be important components of the interaction. In the interaction between Poa ampla and the endophyte Neotyphodium sp., a fungal beta-1,6-glucanase is secreted into the apoplast, and activity of the enzyme is detectable in endophyte-infected plants. Sequence analysis indicates the beta-1,6-glucanase is homologous to enzymes secreted by the mycoparasitic fungi Trichoderma harzianum and Trichoderma virens. DNA gel-blot analysis indicated the beta-1,6-glucanase was encoded by a single gene. As a secreted protein, the beta-1,6-glucanase may have a nutritional role for the fungus. In culture, beta-1,6-glucanase activity was induced in the presence of beta-1,6-glucans. From RNA gel blots, similar beta-1,6-glucanases were expressed in tall fescue (Festuca arundinacea Schreb.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman) infected with the endophyte species Neotyphodium coenophialum and Epichloë festucae, respectively.

Summary CpNpG and CpG methylation was surveyed in a range of vascular and nonvascular plants to determine firstly when CpNpG methylation evolved and secondly whether the two methylation systems found in higher plants were likely to be under common or separate control. Although both systems exist in a wide range of vascular plant taxa, the nonvascular plant taxa appear to contain only CpNpG methylation and this in only very limited amounts. The data suggest that both systems may have evolved at the same time and that speciation involved loss of one or the other methylation system or the evolution of differentiation stage-specific control systems.

When barley (Hordeum vulgare) aleurone layers are subjected to heat shock there is a selective degradation of the normally stable mRNAs encoding secreted proteins. Messages for nonsecreted proteins are not degraded. The synthesis of heat shock proteins is not required for this selective message degradation. Our hypothesis explaining this phenomenon is that a component of the early steps in the synthesis of secreted proteins is damaged by heat shock, resulting in a selective halt in translation on secretory mRNAs, which may in turn lead to degradation of those messages. The first committed step in the synthesis of secreted proteins is the binding of the nascent signal sequence to the signal recognition particle. We have obtained cDNA clones and antibodies for the barley 54-kDa subunit of the signal recognition particle. In cell fractionation experiments, more signal recognition particle was bound to the endoplasmic reticulum membranes and less was in the free particle fraction following a heat shock. The results suggest that heat shock inhibits the release of the signal recognition particle from the endoplasmic reticulum. This would, in turn, inhibit the resumption of translation and may be the underlying cause of the secretory message degradation.

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.

The first step in the routing of newly synthesized proteins into the secretory pathway is the binding of the nascent signal sequence to the signal recognition particle. The mammalian signal recognition particle is a complex consisting of 6 proteins and a single 7S RNA molecule. Signal recognition particle-like complexes have been described from wheat and maize but none of the protein components have yet been described from any plant species. Here we report the cloning and characterization of an Arabidopsis thaliana gene encoding the 54 kDa protein subunit of the signal recognition particle. This is the first report of a SRP-54 sequence for any plant species and the first genomic sequence for any multicellular organism.

Phosphoglycolate phosphatase was partially purified from leaves of Nicotiana rustica using ion exchange and chromatofocusing columns. The native molecular weight of the enzyme was determined to be about 58 kD from Ferguson plots, with a subunit size of about 32 kD. The native enzyme is thus likely to be a dimer. A polyclonal antibody prepared against the LDS denatured enzyme cross reacted with proteins from Nicotiana tabacum, Glycine max, Spinacea oleracea and Arabidopsis thaniana. There was little or no reaction with an Arabidopsis mutant lacking phosphoglycolate phosphatase activity, indicating a much reduced level of phosphoglycolate phosphatase protein in the mutant.

Infection of turf and forage grasses with AcremoniumLink:Fr. and Epichloe(Fr.) Tul. & C. Tul. fungal endophytes is often desirable because it confers enhanced insect tolerance to these plants. Determination of the viability of the endophytes in seed lots is important for commercial producers and consumers of seed. A current method of determining viability relies on staining leaf sheath epidermal peels for the presence of endophyte hyphae. It is difficult to obtain an epidermal peel from a young seedling, so plants must often be grown for several months before checking for viability. The objective of this study was to develop a method of screening very young seedlings for endophyte viability. Here it is reported that endophyte viability could be determined within 2 to 3 wk of germination by staining intact leaf sheath tissue with an alkaline rose bengal stain. This method of endophyte detection allows a more rapid assessment of percentage of viable endophyte in a seed lot.

Strong creeping red fescue (Festuca rubra ssp. rubra) is an important cool season turfgrass species. Cultivars are often infected with the fungal endophyte Epichloè festucae. Endophyte infection is known to confer insect and disease resistance to the plants. The effect of endophyte infection on drought or heat stress tolerance of strong creeping red fescue is not yet established. The objectives of this controlled-environment study were to determine if endophyte infection had any effect on physiological parameters associated with plant tolerance to drought or heat stress or the combination of the two stresses. In this study, endophyte status had no effect on turf quality (TQ), relative water content (RWC), photochemical efficiency, chlorophyll content, electrolyte leakage (EL), or malondialdehyde (MDA) content of the plants under any of the stress treatments. Our results suggested that E. festucae infection had no physiological effects on improving drought, heat or the combined stress tolerance in strong creeping red fescue. [ABSTRACT FROM AUTHOR]