Comparative proteomic analysis provides new insights into the specialization of shoots and stolons in bermudagrass (Cynodon dactylon L.).

BACKGROUND: Bermudagrass (Cynodon dactylon L.) is an important turfgrass species with two types of stems, shoots and stolons. Despite their importance in determining the morphological variance and plasticity of bermudagrass, the intrinsic differences between stolons and shoots are poorly understood. RESULTS: In this study, we compared the proteomes of internode sections of shoots and stolons in the bermudagrass cultivar Yangjiang. The results indicated that 376 protein species were differentially accumulated in the two types of stems. Pathway enrichment analysis revealed that five and nine biochemical pathways were significantly enriched in stolons and shoots, respectively. Specifically, enzymes participating in starch synthesis all preferentially accumulated in stolons, whereas proteins involved in glycolysis and diverse transport processes showed relatively higher abundance in shoots. ADP-glucose pyrophosphorylase (AGPase) and pyruvate kinase (PK), which catalyze rate-limiting steps of starch synthesis and glycolysis, showed high expression levels and enzyme activity in stolons and shoots, respectively, in accordance with the different starch and soluble sugar contents of the two types of stems. CONCLUSIONS: Our study revealed the differences between the shoots and stolons of bermudagrass at the proteome level. The results not only expand our understanding of the specialization of stolons and shoots but also provide clues for the breeding of bermudagrass and other turfgrasses with different plant architectures.

Screening exogenous fibrolytic enzyme preparations for improved in vitro digestibility of bermudagrass haylage.

Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 µg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to the least effective, the most effective EFE at increasing NDFD contained 10 times more endoglucanase III, 17 times more acetylxylan esterase with a cellulose-binding domain 1, 33 times more xylanase III, 25 times more ß-xylosidase, and 7.7 times more polysaccharide monooxygenase with cellulose-binding domain 1 and 3 times more swollenin. The most effective EFE had a much greater quantity of fibrolytic enzymes and key proteins necessary for hemicellulose and lignocellulase deconstruction. This study identified several EFE that increased the NDFD and in vitro fermentation of 4-wk BH and revealed why some EFE are more effective than others.

Effect of the dose of exogenous fibrolytic enzyme preparations on preingestive fiber hydrolysis, ruminal fermentation, and in vitro digestibility of bermudagrass haylage.

Our objectives were to evaluate the effects of the dose rates of 5 Trichoderma reesei and Aspergillus oryzae exogenous fibrolytic enzymes (EFE; 1A, 2A, 11C, 13D, and 15D) on in vitro digestibility, fermentation characteristics, and preingestive hydrolysis of bermudagrass haylage and to identify the optimal dose of each EFE for subsequent in vitro and in vivo studies. In experiment 1, EFE were diluted in citrate-phosphate buffer (pH 6) and applied in quadruplicate in each of 2 runs at 0× (control), 0.5×, 1×, 2×, and 3×; where 1× was the respective manufacturer-recommended dose (2.25, 2.25, 10, 15, and 15g of EFE/kg of dry matter). The suspension was incubated for 24h at 25°C and for a further 24h at 39°C after the addition of ruminal fluid. In experiment 2, a similar approach to that in experiment 1 was used to evaluate simulated preingestive effects, except that sodium azide (0.02% wt/vol) was added to the EFE solution. The suspension was incubated for 24h at 25°C and then 15mL of water was added before filtration to extract water-soluble compounds. For both experiments, data for each enzyme were analyzed separately as a completely randomized block design with a model that included effects of EFE dose, run, and their interaction. In experiment 1, increasing the EFE dose rate nonlinearly increased the DM digestibility of 1A, 2A, 11C, and 13D and the neutral detergent fiber digestibility (NDFD) of 1A, 2A, 11C, and 13D. Optimal doses of 1A, 2A, 11C, 13D, and 15D, as indicated by the greatest increases in NDFD at the lowest dose tested, were 2×, 2×, 1×, 0.5×, and 0.5×, respectively. Increasing the dose rate of 2A, 11C, and 13D nonlinearly increased concentrations of total volatile fatty acids and propionate (mM), decreased their acetate-to-propionate ratios and linearly decreased those of samples treated with 1A and 15D. In experiment 2, increasing the dose rate of each EFE nonlinearly decreased concentrations of netural detergent fiber; also, increasing the dose rate of 1A, 2A, 11C, and 1D nonlinearly increased concentrations of water-soluble carbohydrates and free ferulic acid (µg/g). Application of increasing doses of the EFE increased NDF hydrolysis, NDFD, and ruminal fluid fermentation of bermudagrass haylage, but the optimal dose varied with the EFE.

Physiological responses of somaclonal variants of triploid bermudagrass (Cynodon transvaalensis x Cynodon dactylon) to drought stress.

Eight somaclonal variants with enhanced drought tolerance were isolated from regenerated plants of triploid bermudagrass (Cynodon dactylon x Cynodon transvaalensis cv., TifEagle). Three of them (10-17, 89-02, 117-08) with strong drought tolerance were selected for investigations of physiological responses to drought stress. Compared to the parent control, TifEagle, the somaclonal variants had higher relative water contents and relative growth, and lower ion leakages in the greenhouse tests, while no difference in evapotranspirational water losses and soil water contents was observed between the variants and TifEagle. The variants also had less leaf firing in the field tests under drought stress. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities decreased gradually in responses to drought stress in all plants and exhibited negative correlations with ion leakage, indicating that the declined activities of these antioxidant enzymes were associated with drought injury in the triploid bermudagrass. However, CAT activities were significantly higher in all three variants than in TifEagle during drought stress. Two variants, 10-17 and 89-02, also had significantly higher APX activities than TifEagle before and during the first 4 days of drought treatments. These two lines also showed higher SOD activities after prolonged drought stress. Proline, total soluble sugars and sucrose were accumulated under drought stress in all plants and exhibited positive correlations with ion leakage. More proline and sugars were accumulated in TifEagle than in the variants. The results indicated that higher activities of the antioxidant enzymes in the variants during drought stress are associated with their increased drought tolerance.

Abscisic acid improves drought tolerance of triploid bermudagrass and involves H2O2- and NO-induced antioxidant enzyme activities.

Drought is a major limiting factor for turfgrass growth. Protection of triploid bermudagrass against drought stress by abscisic acid (ABA) and its association with hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) were investigated. ABA treatment increased relative water content, decreased ion leakage and the percentage of dead plants significantly under drought stress. Superoxide dismutase (SOD) and catalase (CAT) activities increased in both ABA-treated and control plants, but more in ABA-treated plants, under drought stress. Malondialdehyde, an indicator of plant lipid peroxidation, was lower in ABA-treated plants than in control plants, indicating that ABA alleviated drought-induced oxidative injury. ABA treatment increased H(2)O(2) and NO contents. ABA-induced SOD and CAT activities could be blocked by scavengers of H(2)O(2) and NO, and inhibitors of H(2)O(2) and NO generation. The results indicated that H(2)O(2) and NO were essential for ABA-induced SOD and CAT activities. Both H(2)O(2) and NO could induce SOD and CAT activities individually. SOD and CAT induced by H(2)O(2) could be blocked by scavenger of NO and inhibitors of NO generation, while SOD and CAT induced by NO could not be blocked by scavenger of H(2)O(2) and inhibitor of H(2)O(2). The results revealed that ABA-induced SOD and CAT activities were mediated sequentially by H(2)O(2) and NO, and NO acted downstream of H(2)O(2).

The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes.

The C4 photosynthetic pathway involves the assimilation of CO2 by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C4 acids. The enzymes of the CO2 concentrating mechanism could be affected under water deficit and limit C4 photosynthesis. Three different C4 grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C4 acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C4 enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of L-malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO2 by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.

[Ecophysiological responses of Festuca arundinacea to high temperature stress].

The measurement of leaf relative water content (RWC), chlorophyll content, cell membrane lipid peroxidation, anti-oxidative system, and photosynthesis of two F. arundinacea cultivars (Barlexas and Crossfire II) and Cynodon dactylon under high temperature (38 degrees C / 30 degrees C, day/ night) showed that with the increasing time of exposure to high temperature, the leaf RWC, chlorophyll content, net photosynthetic rate (P(n)) and photochemical efficiency (F(v)/F(m)) of two F. arundinacea cultivars had a decreasing trend, and the average decrement was smaller for Barlexas than for Crossfire II. After exposed to high temperature for 9 days, the average activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in Barlexas leaves were 19.7%, 17.9% and 17.7% higher than those in Crossfire II leaves, and the P(n) of Barlexas and Crossfire II was decreased by 60.7% and 81.9%, respectively. Under high temperature, the F(v)/F(m) of Barlexas leaves was higher than that of Crossfire II leaves, which could be helpful to mitigate the damage of high temperature to the photosynthetic apparatus of Barlexas. No significant change was observed for each test physiological parameter of C. dactylon leaves with the increase of exposure time under high temperature. The adaptation ability to high temperature was in the order of C. dactylon > Barlexas > Crossfire II.

Real-time quantitative RT-PCR assay of gene expression in plant roots during fungal pathogenesis.

Real-time quantitative RT-PCR is becoming the preferred method for high-sensitivity, rapid-throughput RNA transcript quantification. However, due to the significant developmental costs of dedicated fluorogenic probes, a real-time assay that is simple to establish, comparatively inexpensive, and readily adaptable would be advantageous for the detailed analysis of large sets of expressed sequences. We have devised a flexible real-time quantitative RT-PCR assay that employs a nonspecific DNA binding dye for product detection and uses a relative quantification formula to account for differences in PCR amplification efficiency between the target and reference products. The latter permits the use of an exogenous reference transcript and therefore avoids the normal requirement for the construction of a recombinant RNA reference transcript or extensive characterization of housekeeping gene expression. In an investigation of class II chitinase expression in two varieties of Bermuda grass (Cynodon spp.), following infection with the fungal root pathogen Ophiosphaerella narmari, this assay identified 16- and 28-fold peaks in gene expression at 24 and 96 h after inoculation, respectively.