Transcriptome-Based Weighted Gene Co-Expression Network Analysis Reveals the Photosynthesis Pathway and Hub Genes Involved in Promoting Tiller Growth under Repeated Drought-Rewatering Cycles in Perennial Ryegrass.

Drought stress, which often occurs repeatedly across the world, can cause multiple and long-term effects on plant growth. However, the repeated drought-rewatering effects on plant growth remain uncertain. This study was conducted to determine the effects of drought-rewatering cycles on aboveground growth and explore the underlying mechanisms. Perennial ryegrass plants were subjected to three watering regimes: well-watered control (W), two cycles of drought-rewatering (D2R), and one cycle of drought-rewatering (D1R). The results indicated that the D2R treatment increased the tiller number by 40.9% and accumulated 28.3% more aboveground biomass compared with W; whereas the D1R treatment reduced the tiller number by 23.9% and biomass by 42.2% compared to the W treatment. A time-course transcriptome analysis was performed using crown tissues obtained from plants under D2R and W treatments at 14, 17, 30, and 33 days (d). A total number of 2272 differentially expressed genes (DEGs) were identified. In addition, an in-depth weighted gene co-expression network analysis (WGCNA) was carried out to investigate the relationship between RNA-seq data and tiller number. The results indicated that DEGs were enriched in photosynthesis-related pathways and were further supported by chlorophyll content measurements. Moreover, tiller-development-related hub genes were identified in the D2R treatment, including F-box/LRR-repeat MAX2 homolog (D3), homeobox-leucine zipper protein HOX12-like (HOX12), and putative laccase-17 (LAC17). The consistency of RNA-seq and qRT-PCR data were validated by high Pearson's correlation coefficients ranging from 0.899 to 0.998. This study can provide a new irrigation management strategy that might increase plant biomass with less water consumption. In addition, candidate photosynthesis and hub genes in regulating tiller growth may provide new insights for drought-resistant breeding.

HSFA3 functions as a positive regulator of HSFA2a to enhance thermotolerance in perennial ryegrass.

Perennial ryegrass (Lolium perenne) is a widely used cool season turfgrass with outstanding turf quality and grazing tolerance. High temperature is the key factor restricting the distribution of perennial ryegrass in temperate and sub-tropic regions. In this study, we found that one HEAT SHCOK TRANSCRIPTION FACOTR (HSF) class A gene from perennial ryegrass, LpHSFA3, was highly induced by heat stress. LpHSFA3 is localized in nucleus and functions as a transcription factor. Ectopic overexpression of LpHSFA3 in Arabidopsis improved thermotolerance and rescued heat sensitive deficiency of athsfa3 mutant. Overexpression of LpHSFA3 in perennial ryegrass enhanced heat tolerance and increased survival rate in summer season as evidenced by decreased EL and MDA, increased number of green leaves and total chlorophyll content. LpHSFA3 binds to the HSE region in LpHSFA2a promoter to constitutively activate the expression of LpHSFA2a and downstream heat stress responsive genes. Ectopic overexpression of LpHSFA2a consequently rescued thermal sensitivity of athsfa3 mutant and enhanced thermotolerance of athsfa2 mutant. Perennial ryegrass protoplasts with overexpression of LpHSFA3 and LpHSFA2a exhibited induction of similar subsets of heat responsive genes. These results indicated that transcription factor LpHSFA3 functions as positive regulator of LpHSFA2a to improve thermotolerance of perennial ryegrass, providing further evidence to understand the regulatory networks of plant heat stress response.

Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth-and-differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

The salt-tolerance of perennial ryegrass is linked with root exudate profiles and microflora recruitment.

Salinity poses a significant threat to plant growth and development. The root microbiota plays a key role in plant adaptation to saline environments. Nevertheless, it remains poorly understood whether and how perennial grass plants accumulate specific root-derived bacteria when exposed to salinity. Here, we systematically analyzed the composition and variation of rhizosphere and endophytic bacteria, as well as root exudates in perennial ryegrass differing in salt tolerance grown in unsterilized soils with and without salt. Both salt-sensitive (P1) and salt-tolerant (P2) perennial ryegrass genotypes grew better in unsterilized soils compared to sterilized soils under salt stress. The rhizosphere and endophytic bacteria of both P1 and P2 had lower alpha-diversity under salt treatment compared to control. The reduction of alpha-diversity was more pronounced for P1 than for P2. The specific root-derived bacteria, particularly the genus Pseudomonas, were enriched in rhizosphere and endophytic bacteria under salt stress. Changes in bacterial functionality induced by salt stress differed in P1 and P2. Additionally, more root exudates were altered under salt stress in P2 than in P1. The content of important root exudates, mainly including phenylpropanoids, benzenoids, organic acids, had a significantly positive correlation with the abundance of rhizosphere and endophytic bacteria under salt stress. The results indicate that the interactions between root-derived bacteria and root exudates are crucial for the salt tolerance of perennial ryegrass, which provides a potential strategy to manipulate root microbiome for improved stress tolerance of perennial grass species.

Nitrogen offset potential in a multiyear farmlet-scale study: Milk and herbage production from grazed perennial ryegrass-white clover swards.

The objective of this study was to quantify the farm gate nitrogen (N) offset potential of perennial ryegrass (Lolium perenne L.; PRG) white clover (Trifolium repens L.; WC) swards by comparing the herbage and milk production from dairy farmlets that were simulations of full farming systems. A study was established where 120 cows were randomly assigned to 4 farmlets of 10.9 ha (stocking rate: 2.75 cow/ha), composed of 20 paddocks each. Cows were fed 526 kg of DM of concentrate on average each year. The 4 grazing treatments were PRG-only at 150 or 250 kg of N/ha and PRG-WC at 150 or 250 kg of N/ha. Cows remained in their treatment group for an entire grazing season and were re-randomized as they calved across treatments each year. As cows calved in the spring as standard practice in Ireland, they were rotationally grazed from early February both day and night (weather permitting) to mid-November, to a target postgrazing sward height of 4.0 cm. Mean sward WC content was 18.1% and 15.4% for the 150 and 250 kg of N/ha PRG-WC treatments, respectively over the 3-yr period. When WC was included, lowering the N rate did not reduce pregrazing yield, pregrazing height, or herbage removed, but those factors decreased significantly when WC was absent. Total annual herbage DM production was 13,771, 15,242, 14,721, and 15,667 kg of DM/ha for PRG-only swards receiving 150 or 250 kg of N/ha and PRG-WC swards receiving 150 or 250 kg of N/ha, respectively. In addition, when WC was present, compressed postgrazing sward heights were lower (4.10 vs. 4.21 cm) and herbage allowance (approximately 17 kg/cow feed allocation per cow per day) higher than the high-N control (+ 0.7 kg of DM/cow per day). There was a significant increase in milk production, both per cow and per hectare, when WC was included in PRG swards. Over the 3-yr study, cows grazing PRG-WC had greater milk (+304 kg) and milk solids (+31 kg of fat + protein) yields than cows grazing PRG-only swards. This significant increase in milk production suggests that the inclusion of WC in grazing systems can be effectively used to increase milk production per cow and per hectare and help offset nitrogen use. This result shows the potential to increase farm gate N use efficiency and reduce the N surplus compared with PRG-dominant sward grazing systems receiving 250 kg of N/ha, without negatively affecting milk solids yield or herbage production, thus increasing farm profit by €478/ha.

The Uptake of Rare Trace Elements by Perennial Ryegrass (Lolium perenne L.).

Technological development has increased the use of chemical elements that have hitherto received scant scientific attention as environmental contaminants. Successful management of these rare trace elements (RTEs) requires elucidation of their mobility in the soil-plant system. We aimed to determine the capacity of Lolium perenne (a common pasture species) to tolerate and accumulate the RTEs Be, Ga, In, La, Ce, Nd, and Gd in a fluvial recent soil. Cadmium was used as a reference as a well-studied contaminant that is relatively mobile in the soil-plant system. Soil was spiked with 2.5-283 mg kg-1 of RTE or Cd salts, representing five, 10, 20, and 40 times their background concentrations in soil. For Be, Ce, In, and La, there was no growth reduction, even at the highest soil concentrations (76, 1132, 10.2, and 874 mg kg-1, respectively), which resulted in foliar concentrations of 7.1, 12, 0.11, and 50 mg kg-1, respectively. The maximum no-biomass reduction foliar concentrations for Cd, Gd, Nd, and Ga were 0.061, 0.1, 7.1, and 11 mg kg-1, respectively. Bioaccumulation coefficients ranged from 0.0030-0.95, and increased Ce < In < Nd ≅ Gd < La ≅ Be ≅ Ga < Cd. Beryllium and La were the RTEs most at risk of entering the food chain via L. perenne, as their toxicity thresholds were not reached in the ranges tested, and the bioaccumulation coefficient (plant/soil concentration quotient) trends indicated that uptake would continue to increase at higher soil concentrations. In contrast, In and Ce were the elements least likely to enter the food chain. Further research should repeat the experiments in different soil types or with different plant species to test the robustness of the findings.

The genetic variation in drought resistance in eighteen perennial ryegrass varieties and the underlying adaptation mechanisms.

BACKGROUND: Drought resistance is a complex characteristic closely related to the severity and duration of stress. Perennial ryegrass (Lolium perenne L.) has no distinct drought tolerance but often encounters drought stress seasonally. Although the response of perennial ryegrass to either extreme or moderate drought stress has been investigated, a comprehensive understanding of perennial ryegrass response to both conditions of drought stress is currently lacking. RESULTS: In this study, we investigated the genetic variation in drought resistance in 18 perennial ryegrass varieties under both extreme and moderate drought conditions. The performance of these varieties exhibited obvious diversity, and the survival of perennial ryegrass under severe stress was not equal to good growth under moderate drought stress. 'Sopin', with superior performance under both stress conditions, was the best-performing variety. Transcriptome, physiological, and molecular analyses revealed that 'Sopin' adapted to drought stress through multiple sophisticated mechanisms. Under stress conditions, starch and sugar metabolic enzymes were highly expressed, while CslA was expressed at low levels in 'Sopin', promoting starch degradation and soluble sugar accumulation. The expression and activity of superoxide dismutase were significantly higher in 'Sopin', while the activity of peroxidase was lower, allowing for 'Sopin' to maintain a better balance between maintaining ROS signal transduction and alleviating oxidative damage. Furthermore, drought stress-related transcriptional and posttranscriptional regulatory mechanisms, including the upregulation of transcription factors, kinases, and E3 ubiquitin ligases, facilitate abscisic acid and stress signal transduction. CONCLUSION: Our study provides insights into the resistance of perennial ryegrass to both extreme and moderate droughts and the underlying mechanisms by which perennial ryegrass adapts to drought conditions.

High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission.

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte-grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.

Combined genomic and transcriptomic analysis reveals the contribution of tandem duplication genes to low-temperature adaptation in perennial ryegrass.

Perennial ryegrass (Lolium perenne L.) is an agronomically important cool-season grass species that is widely used as forage for ruminant animal production and cultivated in temperate regions for the establishment of lawns. However, the underlying genetic mechanism of the response of L. perenne to low temperature is still unclear. In the present study, we performed a comprehensive study and identified 3,770 tandem duplication genes (TDGs) in L. perenne, and evolutionary analysis revealed that L. perenne might have undergone a duplication event approximately 7.69 Mya. GO and KEGG pathway functional analyses revealed that these TDGs were mainly enriched in photosynthesis, hormone-mediated signaling pathways and responses to various stresses, suggesting that TDGs contribute to the environmental adaptability of L. perenne. In addition, the expression profile analysis revealed that the expression levels of TDGs were highly conserved and significantly lower than those of all genes in different tissues, while the frequency of differentially expressed genes (DEGs) from TDGs was much higher than that of DEGs from all genes in response to low-temperature stress. Finally, in-depth analysis of the important and expanded gene family indicated that the members of the ELIP subfamily could rapidly respond to low temperature and persistently maintain higher expression levels during all low temperature stress time points, suggesting that ELIPs most likely mediate low temperature responses and help to facilitate adaptation to low temperature in L. perenne. Our results provide evidence for the genetic underpinning of low-temperature adaptation and valuable resources for practical application and genetic improvement for stress resistance in L. perenne.

18 O enrichment of sucrose and photosynthetic and nonphotosynthetic leaf water in a C3 grass-atmospheric drivers and physiological relations.

The 18 O enrichment (Δ18 O) of leaf water affects the Δ18 O of photosynthetic products such as sucrose, generating an isotopic archive of plant function and past climate. However, uncertainty remains as to whether leaf water compartmentation between photosynthetic and nonphotosynthetic tissue affects the relationship between Δ18 O of bulk leaf water (Δ18 OLW ) and leaf sucrose (Δ18 OSucrose ). We grew Lolium perenne (a C3 grass) in mesocosm-scale, replicated experiments with daytime relative humidity (50% or 75%) and CO2 level (200, 400 or 800 µmol mol-1 ) as factors, and determined Δ18 OLW , Δ18 OSucrose and morphophysiological leaf parameters, including transpiration (Eleaf ), stomatal conductance (gs ) and mesophyll conductance to CO2 (gm ). The Δ18 O of photosynthetic medium water (Δ18 OSSW ) was estimated from Δ18 OSucrose and the equilibrium fractionation between water and carbonyl groups (εbio ). Δ18 OSSW was well predicted by theoretical estimates of leaf water at the evaporative site (Δ18 Oe ) with adjustments that correlated with gas exchange parameters (gs or total conductance to CO2 ). Isotopic mass balance and published work indicated that nonphotosynthetic tissue water was a large fraction (~0.53) of bulk leaf water. Δ18 OLW was a poor proxy for Δ18 OSucrose , mainly due to opposite Δ18 O responses of nonphotosynthetic tissue water (Δ18 Onon-SSW ) relative to Δ18 OSSW , driven by atmospheric conditions.

Different LED light intensity and quality change perennial ryegrass (Lolium perenne L.) physiological and growth responses and water and energy consumption.

Light intensity and spectral composition highly affect plant physiology, growth, and development. According to growing conditions, each species and/or cultivar has an optimum light intensity to drive photosynthesis, and different light spectra trigger photosynthetic responses and regulate plant development differently. For the maintenance of natural sports pitches, namely professional football competitions, turf quality is a key condition. Due to the architecture of most football stadiums, the lawns receive low intensities of natural light, so supplementary artificial lighting above the turf is required. The use of light-emitting diodes (LEDs) can have a higher cost-benefit ratio than traditional high-pressure sodium lamps. The continuous emission spectrum, combined with high spectral selectivity and adjustable optical power, can be used to optimize plant growth and development. Thus, perennial ryegrass (Lolium perenne L.) plants, commonly used for lawns, were primarily grown at three different intensities (200, 300, and 400 µmol m-2 s-1) of cool white light. Despite the higher water and energy consumption, 400 µmol m-2 s-1 maximizes the plant's efficiency, with higher photosynthetic rates and foliar pigment concentration, and more foliar soluble sugars and aboveground biomass accumulation. Then, it was evaluated the perennial ryegrass (Double and Capri cultivars) response to different spectral compositions [100% cool white (W), 80% Red:20% Blue (R80:B20), 90% Red:10% Blue (R90:B10), and 65% Red:15% Green:20% Blue (R65:G15:B20)] at 400 µmol m-2 s-1. Both cultivars exhibited similar responses to light treatments. In general, W contributed to the better photosynthetic performance and R90:B10 to the worst one. Water consumption and aboveground biomass were equal in all light treatments. R80:B20 allows energy savings of 24.3% in relation to the W treatment, showing a good compromise between physiological performance and energy consumption.

Methane emission, nutrient digestibility, and rumen microbiota in Holstein heifers fed 14 different grass or clover silages as the sole feed.

This experiment investigated the variation in enteric methane production and associated gas exchange parameters, nutrient digestibility, rumen fermentation, and rumen microbiome when a range of silages based on different forage types (grass or clover), and different species within the 2 types, were fed as the sole feed to heifers. Three grass species (perennial ryegrass, festulolium, and tall fescue) and 2 clover species (red clover and white clover) were included. Perennial ryegrass was harvested at 2 maturity stages in the primary growth, white clover was harvested once in the primary growth, and 4 cuts of festulolium and tall fescue and 3 cuts of red clover were harvested during the growing season, giving 14 different silage batches in total. Sixteen Holstein heifers 16 to 21 mo old and 2 to 5 mo in pregnancy were fed the silages ad libitum as the sole feed in an incomplete crossover design. Each silage was fed to 4 heifers, except for the 2 perennial ryegrass silages, which were fed to 8 heifers; in total 64 observations. The CH4 production was measured for 3 d in respiration chambers. Heifers fed clover silages had higher dry matter intake (DMI) compared with heifers fed grass silages, and heifers fed tall fescue silages had the numerically the lowest DMI. Compared with grass silages, feeding clover silages led to higher crude protein digestibility but lower neutral detergent fiber (NDF) digestibility. Rumen pH was higher in heifers fed clover silages compared with those fed grass silages. Based on composition analysis, the rumen microbiota of the heifers clustered clearly according to forage type and species. More specifically, 7 of the 34 dominating rumen bacterial genus-level groups showed higher relative abundances for the clover silages, whereas 7 genus-level groups showed higher abundances for the grass silages. Methane yield was higher for heifers fed grass silages than for those fed clover silages when methane production was related to dry matter and digestible organic matter intake, whereas the opposite was seen when related to NDF digestion. The gross energy lost as methane (CH4 conversion factor, %) reduced from 7.5% to 6.7%, equivalent to an 11% reduction. The present study gives the outlines for choosing the optimal forage type and forage species with respect to nutrient digestibility and enteric methane emission in ruminants.

Potential of carvacrol as plant growth-promotor and green fungicide against fusarium wilt disease of perennial ryegrass.

Perennial ryegrass (Lolium perenne L.) is a valuable forage and soil stabilisation crop. Perennial crops have long been associated with good environmental performance and ecosystem stability. Vascular wilt diseases caused by Fusarium species are the most damaging plant diseases affecting both woody perennials and annual crops. Therefore, the aim of the present study was the assessment of the preventive and growth-promoting effects of carvacrol against Fusarium oxysporum, F. solani, and F. nivale (phylogenetically analyzed on the basis of internal transcribed spacer (ITS) regions) causing vascular wilt of ryegrass in vitro and under greenhouse conditions. To accomplish this aim, various parameters were monitored including coleoptile development, rhizogenesis, the incidence of coleoptile lesions, disease index, the visual appearance of ryegrass health, ryegrass organic matter and soil fungal load. The results obtained showed that F. nivale was highly harmful to ryegrass seedlings compared to other Fusarium species. Furthermore, carvacrol with 0.1 and 0.2 mg/mL protected significantly the seedlings against Fusarium wilt diseases both in vitro and in the greenhouse. Simultaneously, carvacrol also functioned as a seedling growth promoter, as is reflected in all monitored parameters, such as the recovery of seedling height and root length, and the development of new leaf buds and secondary roots. Carvacrol proved to be effective plant growth promoter and a bio-fungicide against Fusarium vascular diseases.

Fusarium equiseti-inoculation altered rhizosphere soil microbial community, potentially driving perennial ryegrass growth and salt tolerance.

Fusarium equiseti is an effective plant growth-promoting fungi that induce systemic disease resistance in plants. However, the role of F. equiseti in regulating salt stress response and the underlying mechanisms remain largely unknown. Here, we investigated the effect of F. equiseti Z7 strain on the growth and salt stress response in perennial ryegrass. Additionally, the role of Z7 in regulating the abundance, composition, and structure of native microbial communities in the rhizosphere soil was determined. We observed that Z7 could produce indole-3-acetic acid (IAA) and siderophores. Hence, Z7 inoculation further enhanced plant growth and salt tolerance in perennial ryegrass. Inoculating Z7 increased K+ and decreased Na+ in plant tissues. Z7 inoculation also enhanced soil quality by reducing soluble salt and increasing available phosphorus. Moreover, inoculating Z7 altered the compositions of bacterial and fungal communities in the rhizosphere soil. For instance, beneficial bacterial genera, such as Flavobacterium, Enterobacter, Agrobacterium, and Burkholderiales were dominantly enriched in Z7-inoculated soil. Interestingly, the relative abundance of these genera showed significantly positive correlations with the fresh weight of perennial ryegrass. Our results demonstrate that Z7 could remarkably promote plant growth and salt tolerance by regulating ion homeostasis in plant tissues and microbial communities in the rhizosphere soil. This study provides a scientific foundation for applying microbes to improve plant growth under extreme salt stress conditions.

Comparison of milk and grass composition from grazing Irish dairy herds with and without milk fat depression.

BACKGROUND: This study investigated the factors relating to pasture chemical and fatty acid (FA) composition that influence the milk fat percentage of spring calving, grazing dairy cows. The relationship between milk fat percentage and FA composition of the milk in these herds was also investigated. RESULTS: Milk protein percentage, milk casein percentage and cheddar cheese yield were increased in milk from HMF herds. Cows from LMF herds did not have negatively altered milk processability including rennet coagulation time (RCT), pH and ethanol stability. Crude protein, NDF, ADF, ether extract and total FA content of pasture was not different between LMF and HMF herds. Milk fat concentration of conjugated linoleic acid (CLA) t10, c12 was not different between HMF and LMF herds. Pre-grazing herbage mass and pasture content of crude protein, neutral detergent fibre (NDF) and total FA were similar between HMF and LMF herds. Pasture offered to LMF herds had a higher concentration of monounsaturated fatty acids (MUFA). A strong negative relationship (r = -0.40) was evident between milk fat percentage and pasture crude protein content for MMF herds (3.31-3.94% milk fat). CONCLUSIONS: This research reports improved milk protein percentage, milk casein percentage and cheddar cheese yield from HMF herds compared to LMF herds. Milk processability was not impacted by low milk fat percentage. Pasture NDF and total fatty acid content was similar in HMF herds and LMF herds. Milk fat percentage had a strong negative association (r = -0.40) with pasture crude protein content in MMF herds (MF 3.31-3.94%). Correlation values between pasture chemical and FA composition and milk fat percentage in LMF herds and HMF herds were low, indicating that diet is not the only causative factor for variation in milk fat of grazing dairy cows. Comparison of milk fatty acid composition from herds with and without milk fat depression suggests that there may be other fatty acids apart from CLA t10, c12 that contribute to the inhibition of milk fat synthesis during milk fat depression in grazing herds.

Fiber digestibility and protein value of pulp silage for lactating dairy cows: Effects of wet fractionation by screw pressing of perennial ryegrass.

The aim of the study was to investigate the effects of substituting silage of chopped grass with pulp silage of grass fractionated once or twice in a biorefinery using a screw press on fiber kinetics, protein value, and production of CH4 in dairy cows. Six lactating multiparous Holstein cows in mid-lactation (176 ± 93 d in milk), cannulated in the rumen, duodenum, and ileum, were used in an incomplete 6 × 4 Latin square design with a 2 × 3 factorial arrangement of treatments. Perennial ryegrass was harvested in third regrowth from the same field at early and late developmental stage (35 and 44 d of regrowth, respectively) and subjected to 1 of 3 types of processing within each developmental stage. Grass was either harvested for normal silage making (mowed, wilted, chopped, and ensiled), or harvested fresh and fractionated using a screw press. Half of the pulp from the first fractionation was ensiled, whereas the other half of the pulp was rehydrated, fractionated a second time, and pulp hereof was ensiled. The grass and pulp silages were used with concentrates (65:35 forage to concentrate ratio) to make total mixed rations (TMR) based on either silage of chopped grass (GS), pulp silage of grass fractionated once (1×P), or pulp silage of grass fractionated twice (2×P), harvested either at early (E) or late (L) developmental stage resulting in 6 different TMR treatments (EGS, E1×P, E2×P, LGS, L1×P, L2×P). The TMR were fed for ad libitum intake and samples of intestinal digesta and feces were collected for determination of digestibility. The effect of processing on ash-free neutral detergent fiber (aNDFom) concentration in silages depended on developmental stage, but showed that within each developmental stage, pulp silage of grass fractionated twice had higher aNDFom concentration than pulp silage of grass fractionated once and silage of chopped grass. The 2×P resulted in lower (14.9 ± 0.55 vs. 17.5 ± 0.54 kg/d) dry matter intake (DMI) compared with GS. The effects of processing and developmental stage interacted such that apparent total-tract aNDFom digestibility was higher (784 ± 13 vs. 715 ± 13 g/kg) for L2×P compared with LGS, whereas no difference was found between E2×P and EGS. Moreover, the protein value was higher (106 ± 5 vs. 92 ± 5 g AA digested in the small intestine/kg of DMI) for 2×P compared with GS. Unexpectedly, processing had no effect on fractional rate of digestion of digestible aNDFom or CH4 yield (L/kg of DMI), whereas feeding forages harvested at early compared with late developmental stage resulted in lower CH4 yield. Feeding pulp silage of grass fractionated once generally yielded results intermediate to cows fed silage of chopped grass and pulp silage of grass fractionated twice. This study showed that pulp silage of fractionated grass could serve as feed for dairy cows because the fiber digestibility and protein value improved, but further research investigating effects of physical processing of forage on fiber kinetics is required.

Fine-Mapping and Comparative Genomic Analysis Reveal the Gene Composition at the S and Z Self-incompatibility Loci in Grasses.

Self-incompatibility (SI) is a genetic mechanism of hermaphroditic plants to prevent inbreeding after self-pollination. Allogamous Poaceae species exhibit a unique gametophytic SI system controlled by two multi-allelic and independent loci, S and Z. Despite intense research efforts in the last decades, the genes that determine the initial recognition mechanism are yet to be identified. Here, we report the fine-mapping of the Z-locus in perennial ryegrass (Lolium perenne L.) and provide evidence that the pollen and stigma components are determined by two genes encoding DUF247 domain proteins (ZDUF247-I and ZDUF247-II) and the gene sZ, respectively. The pollen and stigma determinants are located side-by-side and were genetically linked in 10,245 individuals of two independent mapping populations segregating for Z. Moreover, they exhibited high allelic diversity as well as tissue-specific gene expression, matching the expected characteristics of SI determinants known from other systems. Revisiting the S-locus using the latest high-quality whole-genome assemblies revealed a similar gene composition and structure as found for Z, supporting the hypothesis of a duplicated origin of the two-locus SI system of grasses. Ultimately, comparative genomic analyses across a wide range of self-compatible and self-incompatible Poaceae species revealed that the absence of a functional copy of at least one of the six putative SI determinants is accompanied by a self-compatible phenotype. Our study provides new insights into the origin and evolution of the unique gametophytic SI system in one of the largest and economically most important plant families.

Humic acids enhance salt stress tolerance associated with pyrroline 5-carboxylate synthetase gene expression and hormonal alteration in perennial ryegrass (Lolium perenne L.).

Humic acid (HA) has been used as an important component in biostimulant formulations to enhance plant tolerance to salt stress, but the mechanisms underlying are not fully understood. This study was to investigate the physiological and molecular mechanisms of HA's impact on salt stress tolerance in perennial ryegrass (Lolium perenne L.). The two types of HA were extracted from weathered coal samples collected from Wutai County (WTH) and Jingle County (JLH) of Shanxi Province, China. The grass seedlings subjected to salt stress (250 mM NaCl) were treated with HA solutions containing 0.01% WTH (W/V) or 0.05% JLH (W/V), respectively. The HA treatments improved leaf photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) and reduced leaf oxidative injury (lower malondialdehyde content) and Pro and intercellular CO2 concentrations in salt-stressed perennial ryegrass. The HA treatments also reversed the decline in antioxidative enzymes ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity and improved growth and anti-senescence hormones indole-3-acetic acid (IAA) and brassinosteroid (BR). The HA treatments reduced the relative expression of P5CS and its downstream products proline (Pro) and the stress defense hormones abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and polyamines (PA). The results of this study indicate that the application of HAs may improve salt stress tolerance by regulating P5CS gene expression related to osmotic adjustment and increasing the activity of antioxidant enzymes and anti-senescence hormones in perennial ryegrass.

Fermentation quality, bacterial community, and predicted functional profiles in silage prepared with alfalfa, perennial ryegrass and their mixture in the karst region.

There is little information regarding the dynamics of fermentation products and the bacterial community in silage prepared with alfalfa (MS), perennial ryegrass (LP), and their mixture in the karst region. In this study, we explored the effects of combining MS with LP in different ratios (100% MS, 70% MS + 30% LP, 50% MS + 50% LP, 30% MS + 70% LP and 100% LP; fresh matter basis) on silage chemical composition, fermentation quality, bacterial communities and predicted functions during the ensiling process. Each treatment was prepared in triplicate and stored at room temperature (22-25°C) for 7, 15, and 45 days. The dry matter (DM) and water-soluble carbohydrate content of the silages increased as the LP proportion in the mixed silage increased; at 45 days, the 70% MS + 30% LP, 50% MS + 50% LP and 30% MS + 70% LP silages contained higher (p < 0.05) CP content than the 100% MS and 100% LP silages. The 30% MS + 70% LP and 100% LP silages exhibited lower (p < 0.05) pH and higher (p < 0.05) LA content than the other silages; at 45 days, none of the silages contained PA or BA. As fermentation proceeded, the abundance of harmful (Enterobacteriaceae and Sphingomonas) and beneficial (Lentilactobacillus, Lactiplantibacillus, Secundilactobacillus, and Levilactobacillus) microorganisms decreased and increased, respectively, as the LP proportion in the mixed silage increased. The predicted functional distribution of microbial communities and metabolic pathways revealed that the 30% MS + 70% LP and 100% LP silages had a stronger capacity for fermentation and a weaker capacity for nitrate reduction than the other silages. Moreover, as the fermentation proceeded, the 30% MS + 70% LP and 100% LP treatments enhanced the functions of "Metabolism," "Genetic information processing" and "Organismal systems" at level 1, the functions of "Amino acid metabolism" and "Nucleotide metabolism" at level 2, and the functions of "Metabolic pathways," "Biosynthesis of secondary metabolites," "Biosynthesis of antibiotics" and "Purine metabolism" at level 3. Thus, adding LP could improve the fermentation quality of MS silage by changing the composition and metabolic function of microbes; furthermore, ensiling 30% alfalfa with 70% ryegrass can produce high-quality silage in the karst region.

Transcriptomic Sequencing Analysis on Key Genes and Pathways Regulating Cadmium (Cd) in Ryegrass (Lolium perenne L.) under Different Cadmium Concentrations

Perennial ryegrass (Lolium perenne L.) is an important forage grass and has the potential to be used in phytoremediation, while little information is available regarding the transcriptome profiling of ryegrass leaves in response to high levels of Cd. To investigate and uncover the physiological responses and gene expression characteristics of perennial ryegrass under Cd stress, a pot experiment was performed to study the transcriptomic profiles of ryegrass with Cd-spiked soils. Transcriptome sequencing and comparative analysis were performed on the Illumina RNA-Seq platform at different concentrations of Cd-treated (0, 50 and 500 mg·kg−1 soil) ryegrass leaves and differentially expressed genes (DEGs) were verified by RT-qPCR. The results show that high concentrations of Cd significantly inhibited the growth of ryegrass, while the lower concentrations (5 and 25 mg·kg−1) showed minor effects. The activity levels of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) increased in Cd-treated ryegrass leaves. We identified 1103 differentially expressed genes (DEGs) and profiled the molecular regulatory pathways of ryegrass leaves with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in response to Cd stress. Cd stress significantly increased the membrane part, the metabolic process, the cellular process and catalytic activity. The numbers of unigenes related to signal transduction mechanisms, post-translational modification, replication, recombination and repair significantly increased. KEGG function annotation and enrichment analysis were performed based on DEGs with different treatments, indicating that the MAPK signaling pathway, the mRNA surveillance pathway and RNA transport were regulated significantly. Taken together, this study explores the effect of Cd stress on the growth physiology and gene level of ryegrass, thus highlighting significance of preventing and controlling heavy metal pollution in the future.