Transporters and phytohormones analysis reveals differential regulation of ryegrass (Lolium perenne L.) in response to cadmium and arsenic stresses.

Cadmium (Cd) and arsenic (As) are two highly toxic heavy metals and metalloids that coexist in many situations posing severe threats to plants. Our investigation was conducted to explore the different regulatory mechanisms of ryegrass (Lolium perenne L.) responding to individual and combined Cd and As stresses in hydroponics. Results showed that the ryegrass well-growth phenotype was not affected by Cd stress of 10 mg·L-1. However, As of 10 mg·L-1 caused rapid water loss, proline surge, and chlorosis in shoots, suggesting that ryegrass was highly sensitive to As. Transcriptomic analysis revealed that the transcription factor LpIRO2 mediated the upregulation of ZIP1 and YSL6 that played an important role in Cd tolerance. We found that the presence of As caused the overexpression of LpSWT12, a process potentially regulated by bHLH14, to mitigate hyperosmolarity. Indoleacetic acid (IAA) and abscisic acid (ABA) contents and expression of their signaling-related genes were significantly affected by As stress rather than Cd. We predict a regulatory network to illustrate the interaction between transporters, transcription factors, and signaling transduction, and explain the antagonism of Cd and As toxicity. This present work provides a research basis for plant protection from Cd and As pollution.

Cytonuclear interplay in auto- and allopolyploids: a multifaceted perspective from the Festuca-Lolium complex.

Restoring cytonuclear stoichiometry is necessary after whole-genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto- and allopolyploids of the Festuca-Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well-established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post-transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Cytokinin or ethylene regulation of heat-induced leaf senescence involving transcriptional modulation of WRKY in perennial ryegrass.

Heat stress is a major abiotic stress for temperate plant species with characteristic symptoms of premature leaf senescence. The objectives of this study were to evaluate the physiological effects of cytokinins (CK) and an ethylene inhibitor, aminoethoxyvinylglycine (AVG) on heat-induced leaf senescence in the temperate perennial grass species, perennial ryegrass (Lolium perenne), and to investigate whether WRKY transcription factors (TFs) could be associated with CK- or ethylene-mediated regulation of heat-induced leaf senescence by exogenously applying CK or AVG to perennial ryegrass. Perennial ryegrass plants foliar-sprayed with 6-benzylaminopurine (6-BA), and AVG exhibited prolonged stay-green phenotypes and a lesser degree of leaf senescence under heat stress (35/30°C), as shown by a decline in electrolyte leakage, malondialdehyde content, hydrogen peroxide, and superoxide content, and increased chlorophyll (Chl) content along with reduced activities of Chl-degrading enzymes (pheophytinase and chlorophyllase) and increased activity of Chl-synthesizing enzyme (porphobilinogen deaminase) due to 6-BA or AVG application. The suppression of heat-induced leaf senescence by 6-BA or AVG treatment corresponded with the upregulation of LpWRKY69 and LpWRKY70. The LpWRKY69 and LpWRKY70 promoters were predicted to share conserved cis-elements potentially recognized by TFs in the CK or ethylene pathways. These results indicate that LpWRKY69 and LpWRKY70 may negatively regulate heat-induced leaf senescence through CK or ethylene pathways, conferring heat tolerance in perennial ryegrass.

Redox status upon herbicides application in the control of Lolium multiflorum (2n and 4n) as weed.

Lolium multiflorum Lam. is a winter weed of difficult control found as diploid (2n) and tetraploid plants (4n). Our study aimed to evaluate the responses of antioxidant enzymes and lipid peroxidation, in both diploid and tetraploid ryegrass varieties. Treatments consisted of control plants (without any herbicide application), and four herbicides with different mechanisms of action. Leaf material was collected 36 h after treatment imposition to determine the lipid peroxidation by ferrous oxidation-xylenol (FOX) content, and the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione-S-transferase (GST), and δ-aminolevulinic acid dehydratase (ALAD). Both ryegrass varieties showed oxidative stress mainly due to a downregulated decreased (>31%) in SOD activity and an increase (>32%) in lipid peroxidation (FOX), mainly in ryegrass genotypes exposed to haloxyfop, glyphosate, and iodosulfuron. On the other hand, clethodim-treated plants had an increase in SOD and APX activities, associated with a reduced ALAD activity in both 2n (32%) and 4n (11%) genotypes. In general, the 2n genotype was more affected than the 4n genotype.

Inbred progeny of tall fescue infected with Epichloë fungal endophyte have a high defence compound content and no depression in seed set.

Substantial evidence is available on the capacity of particular strains of Epichloë fungal endophyte to remove the barriers of self-pollination in host grasses. We hypothesized that this might open up new methods to obtain inbred lines for specific turf hybrids and genetic studies. In the present study, we evaluated the first generation of self-pollination derived plants of endophyte infected clones (EIS1 ) from putative genotypes 75B+ and 75C+ and those from the first generation of self-pollination in six commercial tall fescue clones plus their hybrids with 75B- and 75C- for growth, seed yield and polyphenolic content as an index for biosynthesis of defence compounds under field conditions. The results showed that EIS1 had high hyphal density within leaf sheaths and higher growth and seed-related traits in at least one genotype. There were higher amounts of flavonoid and phenolic compounds (up to twofold) in both genotypes than in their hybrid counterparts and endophyte-free progeny. Selected genotypes within EIS1 contained significantly more chlorogenic acid, p-coumaric acid and rutin than the best non-infected genotypes. We conclude that phenotypic selection of individuals from the S1 population is feasible for improving fitness and stress resistance in novel inbred lines of tall fescue for development of new turf cultivars with the desired ecophysiological traits.

Physiological and Gene Expression Responses of Six Annual Ryegrass Cultivars to Cobalt, Lead, and Nickel Stresses.

Heavy metals negatively affect soil quality and crop growth. In this study, we compared the tolerance of six ryegrass cultivars to cobalt (Co2+), lead (Pb2+), and nickel (Ni2+) stresses by analyzing their physiological indexes and transcript levels of genes encoding metal transporters. Compared with the other cultivars, the cultivar Lm1 showed higher germination rates and better growth under Co2+, Pb2+, or Ni2+ treatments. After 48 h of Co2+ treatment, the total antioxidant capacity of all six ryegrass cultivars was significantly increased, especially that of Lm1. In contrast, under Pb2+ stress, total antioxidant capacity of five cultivars was significantly decreased, but that of Lm1 was unaffected at 24 h. Staining with Evans blue dye showed that the roots of Lm1 were less injured than were roots of the other five ryegrass cultivars by Co2+, Pb2+, and Ni2+. Lm1 translocated and accumulated lesser Co2+, Pb2+, and Ni2+ than other cultivars. In Lm1, genes encoding heavy metal transporters were differentially expressed between the shoots and roots in response to Co2+, Pb2+, and Ni2+. The aim of these researches could help find potential resource for phytoremediation of heavy metal contamination soil. The identified genes related to resistance will be useful targets for molecular breeding.

Reciprocal allopolyploid grasses (Festuca × Lolium) display stable patterns of genome dominance.

Allopolyploidization entailing the merger of two distinct genomes in a single hybrid organism, is an important process in plant evolution and a valuable tool in breeding programs. Newly established hybrids often experience massive genomic perturbations, including karyotype reshuffling and gene expression modifications. These phenomena may be asymmetric with respect to the two progenitors, with one of the parental genomes being "dominant." Such "genome dominance" can manifest in several ways, including biased homoeolog gene expression and expression level dominance. Here we employed a k-mer-based approach to study gene expression in reciprocal Festuca pratensis Huds. × Lolium multiflorum Lam. allopolyploid grasses. Our study revealed significantly more genes where expression mimicked that of the Lolium parent compared with the Festuca parent. This genome dominance was heritable to successive generation and its direction was only slightly modified by environmental conditions and plant age. Our results suggest that Lolium genome dominance was at least partially caused by its more efficient trans-acting gene expression regulatory factors. Unraveling the mechanisms responsible for propagation of parent-specific traits in hybrid crops contributes to our understanding of allopolyploid genome evolution and opens a way to targeted breeding strategies.

Gene expression and morphological responses of Lolium perenne L. exposed to cadmium (Cd2+) and mercury (Hg2+).

Soil contamination with heavy metals is a major problem worldwide, due to the increasing impact mainly caused by anthropogenic activities. This research evaluated the phytoremediation capacity of, Lolium perenne for heavy metals such as cadmium (Cd2+) and mercury (Hg2+), and the effects of these metals on morphology, biomass production, and the changes on gene expression. Seeds of L. perenne were exposed to six concentrations of Cd2+ and Hg2+ in the range of 0 to 25 mg L-1, and two mixtures of Cd2+-Hg2. The Non-Observed Effect Level (NOEL) was established with dose response curves and the expression of specific genes was evaluated applying a commercially available quantitative reverse transcription (RT-qPCR) assay. There was no significant effect when exposing the seeds to Hg2+, for Cd2+ the maximum concentration was established in 0.1 mg L-1, and for the two concentrations of mixtures, there was a negative effect. An increase of expression of genes that regulate antioxidant activity and stress was found when the plant was exposed to heavy metals. Given the high tolerance to metals analyzed that was reflected both, the development of the plant and in its molecular response, these results highlight that L. perenne is a plant with phytoremediator potential.

NOL-mediated functional stay-green traits in perennial ryegrass (Lolium perenne L.) involving multifaceted molecular factors and metabolic pathways regulating leaf senescence.

Loss of chlorophyll (Chl) is a hallmark of leaf senescence, which may be regulated by Chl catabolic genes, including NON-YELLOW COLORING 1 (NYC1)-like (NOL). The objective of this study was to determine molecular factors and metabolic pathways underlying NOL regulation of leaf senescence in perennial grass species. LpNOL was cloned from perennial ryegrass (Lolium perenne L.) and found to be highly expressed in senescent leaves. Transient overexpression of LpNOL accelerated leaf senescence and Chl b degradation in Nicotiana benthamiana. LpNOL RNA interference (NOLi) in perennial ryegrass not only significantly blocked Chl degradation in senescent leaves, but also delayed initiation and progression of leaf senescence. This study found that NOL, in addition to functioning as a Chl b reductase, could enact the functional stay-green phenotype in perennial grass species, as manifested by increased photosynthetic activities in NOLi plants. Comparative transcriptomic analysis revealed that NOL-mediated functional stay-green in perennial ryegrass was mainly achieved through the modulation of Chl catabolism, light harvesting for photosynthesis, photorespiration, cytochrome respiration, carbohydrate catabolism, oxidative detoxification, and abscisic acid biosynthesis and signaling pathways.

Cadmium stress suppresses the tillering of perennial ryegrass and is associated with the transcriptional regulation of genes controlling axillary bud outgrowth.

Perennial ryegrass (Lolium perenne L.), a grass species with superior tillering capacity, plays a potential role in the phytoremediation of cadmium (Cd)-contaminated soils. Tiller production is inhibited in response to serious Cd stress. However, the regulatory mechanism of Cd stress-induced inhibition of tiller development is not well documented. To address this issue, we investigated the phenotype, the expression levels of genes involved in axillary bud initiation and bud outgrowth, and endogenous hormone biosynthesis and signaling pathways in seedlings of perennial ryegrass under Cd stress. The results showed that the number of tillers and axillary buds in the Cd-treated seedlings decreased by 67% and 21%, respectively. The suppression of tiller production in the Cd-treated seedlings was more closely associated with the inhibition of axillary bud outgrowth than with bud initiation. Cd stress upregulated the expression level of genes related to axillary bud dormancy and downregulated bud activity genes. Additionally, genes involved in strigolactone biosynthesis and signaling, auxin transport and signaling, and cytokinin degradation were upregulated in Cd-treated seedlings, and cytokinin biosynthesis gene expression were decreased by Cd stress. The content of zeatin in the Cd-treated pants was significantly reduced by 69~85% compared to the control plants. The content of indole-3-acetic acid (IAA) remains constant under Cd stress. Overall, Cd stress induced axillary bud dormancy and subsequently inhibited axillary bud outgrowth. The decrease of zeatin content and upregulation of genes involved in strigolactone signaling and bud dormancy might be responsible for the inhibition of axillary bud outgrowth.

Comparison of cadmium uptake and transcriptional responses in roots reveal key transcripts from high and low-cadmium tolerance ryegrass cultivars.

Cadmium (Cd), which seriously affects plant growth and crop production, is harmful to humans. Previous studies revealed ryegrass (Lolium multiflorum Lam.) exhibits Cd tolerance, and may be useful as a potential hyperaccumulator because of its wide distribution. In this study, the physiological and transcriptional responses of two ryegrass cultivars [i.e., high (LmHC) and low (LmLC) Cd tolerance] to Cd stress were investigated and compared. The Cd tolerance of LmHC was greater than that of LmLC at various Cd concentrations. The uptake of Evans blue dye revealed that Cd-induced root cell mortality was higher in LmLC than in LmHC after a 12-h Cd treatment. Furthermore, the content and influx rate of Cd in LmLC roots were greater than in LmHC roots under Cd stress conditions. The RNA sequencing and quantitative real-time PCR data indicated that the Cd transport regulatory genes (ABCG37, ABCB4, NRAMP4, and HMA5) were differentially expressed between the LmLC and LmHC roots. This expression-level diversity may contribute to the differences in the Cd accumulation and translocation between LmLC and LmHC. These findings may help clarify the physiological and molecular mechanisms underlying ryegrass responses to Cd toxicity. Additionally, ryegrass may be able to hyperaccumulate toxic heavy metals during the phytoremediation of contaminated soil.

Full-Length Transcriptome Assembly of Italian Ryegrass Root Integrated with RNA-Seq to Identify Genes in Response to Plant Cadmium Stress.

Cadmium (Cd) is a toxic heavy metal element. It is relatively easily absorbed by plants and enters the food chain, resulting in human exposure to Cd. Italian ryegrass (Lolium multiflorum Lam.), an important forage cultivated widely in temperate regions worldwide, has the potential to be used in phytoremediation. However, genes regulating Cd translocation and accumulation in this species are not fully understood. Here, we optimized PacBio ISO-seq and integrated it with RNA-seq to construct a de novo full-length transcriptomic database for an un-sequenced autotetraploid species. With the database, we identified 2367 differentially expressed genes (DEGs) and profiled the molecular regulatory pathways of Italian ryegrass with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in response to Cd stress. Overexpression of a DEG LmAUX1 in Arabidopsis thaliana significantly enhanced plant Cd concentration. We also unveiled the complexity of alternative splicing (AS) with a genome-free strategy. We reconstructed full-length UniTransModels using the reference transcriptome, and 29.76% of full-length models had more than one isoform. Taken together, the results enhanced our understanding of the genetic diversity and complexity of Italian ryegrass under Cd stress and provided valuable genetic resources for its gene identification and molecular breeding.

In vitro gas production and rumen fermentation profile of fresh and ensiled genetically modified high-metabolizable energy ryegrass.

We previously generated a high-metabolizable energy (HME) perennial ryegrass (Lolium perenne) by genetically modifying the plant to increase the leaf lipid content. Although substantial progress has been made toward characterizing physiological changes of HME ryegrass, very limited information exists for feeding value and its suitability for adoption into the pastoral system. In this study, independent HME ryegrass lines with a range of elevated leaf lipid concentrations were analyzed for changes in fatty acids and possible associated changes in the broader nutritional profile, including the gross energy, which was found to increase by 6.8%. Because ryegrass is often ensiled and fermentation in the rumen leads to biohydrogenation of fatty acids as well as enteric methane production, we sought to investigate these effects on HME ryegrass. This was achieved by performing mini-scale silos and using an automated gas measurement system to incubate the material in rumen fluid in vitro for 24 h. Our study included treatments comprising 3 independent HME ryegrass genotypes and wild-type control materials prepared fresh and as silage, employing in total 5 incubation studies, using rumen fluids collected from 4 nonlactating Jersey × Holstein cows. At intervals during the incubation, the production of gases, volatile fatty acids, and the degree of biohydrogenation were measured. Statistical data analysis indicated that differences in the nutritional compositions of the ensiled materials largely reflected those of their fresh counterparts. Incubation of both fresh and ensiled HME ryegrass in rumen fluid resulted in: (1) a greater percentage of valuable unsaturated fatty acids compared with the control; (2) a significant reduction of butyrate; and (3) a 10 to 15% decrease in the methane proportion of the total gas production. We conclude that ensiling could be a convenient option for preserving HME as a locally produced high-value supplementary feed; however, large-scale application needs to be investigated. In this paper we discuss the potential use of HME ryegrass to enhancing forage feeding value and the potential environmental benefits to the pastoral agriculture industry.

Cytogenetic and molecular genotyping in the allotetraploid Festuca pratensis × Lolium perenne hybrids.

BACKGROUND: Species of the Festuca and Lolium genera, as well as intergeneric Festuca × Lolium (Festulolium) hybrids, are valuable fodder and turf grasses for agricultural and amenity purposes worldwide. Festulolium hybrids can merge in their genomes agronomically important characteristics. However, in polyploid plants, especially in allopolyploids, the hybridization of divergent genomes could contribute to various abnormalities, such as variability in chromosome number, structural rearrangements, and/or disorders in inheritance patterns. Here we studied these issues in allotetraploid Festuca pratensis × Lolium perenne hybrids. RESULTS: Cytogenetic procedures, including fluorescent in situ hybridization, genomic in situ hybridization, and molecular markers - inter-simple sequence repeats (ISSR) were exploited. This cytogenetic approach indicated the dynamics in the number and distribution of ribosomal RNA genes and structural rearrangements for both parental genomes (Festuca and Lolium) in hybrid karyotypes. The separate analysis of F. pratensis and L. perenne chromosomes in hybrid plants (F2-F3 generations of F. pratensis × L. perenne) revealed the asymmetrical level of rearrangements. Recognized structural changes were mainly located in the distal part of chromosome arms, and in chromosomes bearing ribosomal DNA, they were more frequently mapped in arms without this sequence. Based on the ISSR markers distribution, we found that the tetrasomic type of inheritance was characteristic for the majority of ISSR loci, but the disomic type was also observed. Nonetheless, no preference in the transmission of either Festuca or Lolium alleles to the following generations of allotetraploid F. pratensis × L. perenne hybrid was observed. CONCLUSION: Our study reports cytogenetic and molecular genotyping of the F. pratensis × L. perenne hybrid and its following F2-F3 progenies. The analysis of 137 allotetraploid F. pratensis × L. perenne hybrids revealed the higher level of recombination in chromosomes derived from F. pratensis genome. The results of ISSR markers indicated a mixed model of inheritance, which may be characteristic for these hybrids.

Pyroxasulfone resistance in Lolium rigidum is metabolism-based.

The evolution of resistant weed populations in response to intensive herbicide selection pressure is a global issue. Resistance to post-emergence herbicides is widespread, whereas soil-applied pre-emergence herbicides can often remain effective. For example, in Australia pyroxasulfone is a new pre-emergence soil-applied herbicide which provides control of Lolium rigidum populations resistant to multiple post-emergence herbicide modes of action. A fundamental knowledge of the genetic basis of metabolic resistance in weeds is important for understanding plant evolution pathways under herbicide selection and sustaining long-term weed resistance management. In this study we define the mechanistic basis of resistance to pyroxasulfone in a L. rigidum population. TLC provides evidence that pyroxasulfone resistance is metabolism-based with approximately 88% of parental [14C]-labelled pyroxasulfone metabolized in resistant plants 24 h after the herbicide treatment. HPLC-MS allowed identification of several metabolites of pyroxasulfone formed via a glutathione (GSH) conjugation pathway in pyroxasulfone-resistant L. rigidum plants. However, the initial pyroxasulfone-glutathione conjugate was not found likely due to its labile nature. The observed constitutive over-expression from six to nine-fold of two putative resistance-endowing GST genes was associated with the pyroxasulfone resistance phenotype. The most logical conclusion, based on the data thus far available, is that rapid detoxification of pyroxasulfone mediates pyroxasulfone resistance in L. rigidum plants. Future research is warranted to confirm the hypothesis advanced by this study of rapid pyroxasulfone metabolism due to GSH conjugation mediated by GST over-expressed in pyroxasulfone-resistant plants which similarly leads to the production of distinctive GSH-pyroxasulfone metabolites in L. rigidum and wheat.

B chromosomes are associated with redistribution of genetic recombination towards lower recombination chromosomal regions in perennial ryegrass.

Supernumerary 'B' chromosomes are non-essential components of the genome present in a range of plant and animal species-including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT (Diversity Arrays Technology) marker genetic maps for six perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progeny and their parents, we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared with zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, namely pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders.

Functional characterization and hormonal regulation of the PHEOPHYTINASE gene LpPPH controlling leaf senescence in perennial ryegrass.

Chlorophyll (Chl) degradation occurs naturally during leaf maturation and senescence, and can be induced by stresses, both processes involving the regulation of plant hormones. The objective of this study was to determine the functional roles and hormonal regulation of a gene encoding pheophytin pheophorbide hydrolyase (PPH) that catabolizes Chl degradation during leaf senescence in perennial grass species. A PPH gene, LpPPH, was cloned from perennial ryegrass (Lolium perenne L.). LpPPH was localized in the chloroplast. Overexpressing LpPPH accelerated Chl degradation in wild tobacco, and rescued the stay-green phenotype of the Arabidopsis pph null mutant. The expression level of LpPPH was positively related to the extent of leaf senescence. Exogenous application of abscisic acid (ABA) and ethephon (an ethylene-releasing agent) accelerated the decline in Chl content in leaves of perennial ryegrass, whereas cytokinin (CK) and aminoethoxyvinylglycine (AVG; an ethylene biosynthesis inhibitor) treatments suppressed leaf senescence, corresponding to the up- or down-regulation of LpPPH expression. The promoters of five orthologous PPH genes were predicted to share conserved cis-elements potentially recognized by transcription factors in the ABA and CK pathways. Taken together, the results suggested that LpPPH-mediated Chl breakdown could be regulated positively by ABA and ethylene, and negatively by CK, and LpPPH could be a direct downstream target gene of transcription factors in the ABA and CK signaling pathways.

Silicon-Induced Systemic Defense Responses in Perennial Ryegrass Against Infection by Magnaporthe oryzae.

Sustainable integrated disease management for gray leaf spot of perennial ryegrass may involve use of plant defense elicitors with compatible traditional fungicides to reduce disease incidence and severity. Silicon (Si) has previously been identified as a potential inducer or modulator of plant defenses against different fungal pathogens. To this end, perennial ryegrass was inoculated with the causal agent of gray leaf spot, Magnaporthe oryzae, when grown in soil that was nonamended or amended with three different levels of calcium silicate (1, 5, or 10 metric tons [t]/ha). When applied at a rate of 5 t/ha, calcium silicate was found to significantly suppress gray leaf spot in perennial ryegrass, including a significant reduction of disease incidence (39.5%) and disease severity (47.3%). Additional studies observed nonpenetrated papillae or cell-wall appositions harboring callose, phenolic autofluorogens, and lignin-associated polyphenolic compounds in grass grown in the Si-amended soil. Regarding defense-associated enzyme levels, only following infection did grass grown in Si-amended soil exhibit greater activities of peroxidase and polyphenol oxidase than equivalent inoculated control plants. Also following infection with M. oryzae, grass levels of several phenolic acids, including chlorogenic acid and flavonoids, and relative expression levels of genes encoding phenylalanine ammonia lyase (PALa and PALb) and lipoxygenase (LOXa) significantly increased in Si-amended plants compared with that of nonamended control plants. These results suggest that Si-mediated increase of host defense responses to fungal pathogens in perennial ryegrass has a great potential to be part of an effective integrated disease management strategy against gray leaf spot development.

Evidence for alternative splicing mechanisms in meadow fescue (Festuca pratensis) and perennial ryegrass (Lolium perenne) Rubisco activase gene.

Rubisco activase is required to regulate the catalytic activity of Rubisco in plants, in an ATP-dependent manner. One or two Rubisco activase proteins have been identified in different plant species. In some species, the two isoforms are the products of alternative splicing of the Rubisco activase gene. The aim of this study was to confirm that Lolium perenne and Festuca pratensis plants have two isoforms of Rubisco activase and that they are the products of alternative splicing of common pre-mRNA. Protein gel blot analyses indicated that L. perenne and F. pratensis leaves contained two Rubisco activase proteins. Sequence analysis of cDNA and genomic DNA showed that differential splicing generated two mRNAs that differed in sequence only in the inclusion of 48 bp. The insertion contains a stop codon leading to the synthesis of a shorter polypeptide. Under the conditions of our experiment, the shorter splicing variant of L. perenne and F. pratensis Rubisco activase gene was preferentially produced. Any further studies concerning Rubisco activase genes in L. perenne and/or F. pratensis plants should take into consideration the mechanism of its expression.