Whole-genome sequencing of allotetraploid bermudagrass reveals the origin of Cynodon and candidate genes for salt tolerance.

Bermudagrass (Cynodon dactylon) is a globally distributed, extensively used warm-season turf and forage grass with high tolerance to salinity and drought stress in alkaline environments. However, the origin of the species and genetic mechanisms for salinity tolerance in the species are basically unknown. Accordingly, we set out to study evolution divergence events in the Cynodon genome and to identify genes for salinity tolerance. We developed a 604.0 Mb chromosome-level polyploid genome sequence for bermudagrass 'A12359' (n = 18). The C. dactylon genome comprises 2 complete sets of homoeologous chromosomes, each with approximately 30 000 genes, and most genes are conserved as syntenic pairs. Phylogenetic study showed that the initial Cynodon species diverged from Oropetium thomaeum approximately 19.7-25.4 million years ago (Mya), the A and B subgenomes of C. dactylon diverged approximately 6.3-9.1 Mya, and the bermudagrass polyploidization event occurred 1.5 Mya on the African continent. Moreover, we identified 82 candidate genes associated with seven agronomic traits using a genome-wide association study, and three single-nucleotide polymorphisms were strongly associated with three salt resistance genes: RAP2-2, CNG channels, and F14D7.1. These genes may be associated with enhanced bermudagrass salt tolerance. These bermudagrass genomic resources, when integrated, may provide fundamental insights into evolution of diploid and tetraploid genomes and enhance the efficacy of comparative genomics in studying salt tolerance in Cynodon.

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses.

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.

Transcriptome-wide identification of MAPKKK genes in bermudagrass (Cynodon dactylon L.) and their potential roles in low temperature stress responses.

As upstream components of MAPK cascades, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascades. MAPK cascades are universal modules of signal transduction in eukaryotic organisms and play crucial roles in plant development processes and in responses to biotic and abiotic stress and signal transduction. Members of the MAPKKK gene family have been identified in several plants,however, MAPKKKs have not been systematically studied in bermudagrass (Cynodon dactylon L.). In this study, 55 potential CdMAPKKKs were produced from bermudagrass transcriptome data, of which 13 belonged to the MEKK, 38 to the Raf, and 4 to the ZIK subfamily. Multiple alignment and conserved motif analysis of CdMAPKKKs supported the evolutionary relationships inferred from phylogenetic analyses. Moreover, the distribution pattern in Poaceae species indicated that members of the MAPKKK family were conserved among almost all diploid species, and species-specific polyploidy or higher duplication ratios resulted in an expansion of the MAPKKK family. In addition, 714 co-functional links which were significantly enriched in signal transduction, responses to temperature stimuli, and other important biological processes of 55 CdMAPKKKs were identified using co-functional gene networks analysis; 30 and 19 co-functional genes involved in response to cold or heat stress, respectively, were also identified. Results of promoter analyses, and interaction network investigation of all CdMAPKKKs based on the rice homologs suggested that CdMAPKKKs are commonly associated with regulation of numerous biological processes. Furthermore, 12 and 13 CdMAPKKKs were significantly up- and downregulated, respectively, in response to low temperature stress; among them, six CdMAPKKKs were significantly induced by low temperature stress, at least at one point in time. This is the first study to conduct identification and functional analysis of the MAPKKK gene family in bermudagrass, and our results provide a foundation for further research on the functions of CdMAPKKKs in response to low temperature stress.

The ethylene responsive factor CdERF1 from bermudagrass (Cynodon dactylon) positively regulates cold tolerance.

Cold stress is one of the major environmental factors that limit growth and utilization of bermudagrass [Cynodon dactylon (L.) Pers], a prominent warm-season turfgrass. However, the molecular mechanism of cold response in bermudagrass remains largely unknown. In this study, we characterized a cold-responsive ERF (ethylene responsive factor) transcription factor, CdERF1, from bermudagrass. CdERF1 expression was induced by cold, drought and salinity stresses. The CdERF1 protein was nucleus-localized and encompassed transcriptional activation activity. Transgenic Arabidopsis plants overexpressing CdERF1 showed enhanced cold tolerance, whereas CdERF1-underexpressing bermudagrass plants via virus induced gene silencing (VIGS) method exhibited reduced cold resistance compared with control, respectively. Under cold stress, electrolyte leakage (EL), malondialdehyde (MDA), H2O2 and O2- contents were reduced, while the activities of SOD and POD were elevated in transgenic Arabidopsis. By contrast, these above physiological indicators in CdERF1-underexpressing bermudagrass exhibited the opposite trend. To further explore the possible molecular mechanism of bermudagrass cold stress response, the RNA-Seq analyses were performed. The result indicated that overexpression of CdERF1 activated a subset of stress-related genes in transgenic Arabidopsis, such as CBF2, pEARLI1 (lipid transfer protein), PER71 (peroxidase) and LTP (lipid transfer protein). Interestingly, under-expression of CdERF1 suppressed the transcription of many genes in CdERF1-underexpressing bermudagrass, also including pEARLI1 (lipid transfer protein) and PER70 (peroxidase). All these results revealed that CdERF1 positively regulates plant cold response probably by activating stress-related genes, PODs, CBF2 and LTPs. This study also suggests that CdERF1 may be an ideal candidate in the effort to improve cold tolerance of bermudagrass in the further molecular breeding.

Cadmium Tolerance of Perennial Ryegrass Induced by Aspergillus aculeatus.

Cadmium (Cd) pollution is becoming increasingly prevalent, posing a global environmental hazard due to its negative effects on plants growth and human health. Phytoremediation is a green technology that involves uptake of Cd from the soil by a combination of plants and associated microbes. The objective of this study was to investigate the role of Aspergillus aculeatus in perennial ryegrass Cd tolerance. This fungus produced indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylate deaminase. Physiological traits including growth rate, turf quality and chlorophyll content were measured to evaluate the physiological responses of perennial ryegrass to Cd stress. These physiological traits were improved after inoculated with A. aculeatus. Inoculation of A. aculeatus actively reduced DTPA-Cd concentration in the soil and Cd translocation to plant shoots. Chlorophyll a fluorescence transient and the C/N ratio in shoots were elevated by A. aculeatus, which implied that the fungus could protect the photosystem II against Cd stress and increase the photosynthetic efficiency. These results suggested that A. aculeatus is beneficial in improving Cd tolerance of perennial ryegrass and reducing Cd-induced injuries, thus, it has promising potential for application of phytostabilization in Cd contaminated soil.

Effects of cadmium-resistant fungi Aspergillus aculeatus on metabolic profiles of bermudagrass [Cynodondactylon (L.)Pers.] under Cd stress.

Plants' tolerance to heavy metal stress may be induced by the exploitation of microbes. The objectives of this study were to investigate the effect of cadmium (Cd)-resistant fungus, Aspergillus aculeatus, on tolerance to Cd and alteration of metabolites in bermudagrass under Cd stress, and identify the predominant metabolites associated with Cd tolerance. Two genotypes of bermudagrass with contrasting Cd tolerance (Cd-sensitive 'WB92' and Cd-tolerant 'WB242') were exposed to 0, 50, 150 and 250 mg kg-1 Cd for 21 days. Physiological responses of bermudagrass to Cd stress were evaluated based on the relative growth rate (RGR) and normalized relative transpiration rate (NRT). Plants inoculated with A. aculeatus exhibited higher RGR and NRT under Cd stress than those of non-inoculated plants, regardless of genotypes. A total of 32 Cd-responsive metabolites in leaves and 21 in roots were identified in the two genotypes, including organic acids, amino acids, sugars, and fatty acids and others. Interestingly, under Cd stress, the leaves of inoculated 'WB92' accumulated less citric acid, aspartic acid, glutamic acid, sucrose, galactose, but more sorbose and glucose, while inoculated 'WB242' leaves had less citric acid, malic acid, sucrose, sorbose, but more fructose and glucose, compared to non-inoculated plants. In 'WB92' roots, the A. aculeatus reduced mannose content, but increased trehalose and citric acid content, while in 'WB242', it decreased sucrose, but enhanced citric acid content, compared to Cd regime. The results of this study suggest that A. aculeatus may induce accumulation of different metabolites associated with Cd tolerance in bermudagrass.

Amelioration of Salt Stress on Bermudagrass by the Fungus Aspergillus aculeatus.

There is considerable evidence that plant abiotic-stress tolerance can be evoked by the exploitation of a globally abundant microbe. A. aculeatus, which was initially isolated from the rhizosphere of bermudagrass, has been shown to increase heavy metal tolerance in turfgrasses. Here, we report on the potential of A. aculeatus to induce tolerance to salt stress in bermudagrass. Physiological markers for salt stress, such as plant growth rate, lipid peroxidation, photosynthesis, and ionic homeostasis were assessed. Results indicated that strain A. aculeatus produced indole-3-acetic acid (IAA) and siderophores and exhibited a greater capacity for Na+ absorption under salt stress. The plant inoculation by A. aculeatus increased plant growth and attenuated the NaCl-induced lipid peroxidation in roots and leaves of bermudagrass. The fungus significantly elevated the amount of IAA and glutathione and slightly enhanced photosynthetic efficiency of salt-treated bermudagrass. Tissues of inoculated plants had significantly increased concentrations of K+ but lower Na+ concentrations than those of uninoculated regimes. It appears that the role of A. aculeatus in alleviating bermudagrass salt stress is partly to produce IAA, to increase the activity of antioxidases, to absorb Na+ by fungal hyphae, and to prevent the plant from ionic homeostasis disruption.

Effects of ethylene on photosystem II and antioxidant enzyme activity in Bermuda grass under low temperature.

The phytohormone ethylene has been reported to mediate plant response to cold stress. However, it is still debated whether the effect of ethylene on plant response to cold stress is negative or positive. The objective of the present study was to explore the role of ethylene in the cold resistance of Bermuda grass (Cynodon dactylon (L).Pers.). Under control (warm) condition, there was no obvious effect of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the antagonist Ag(+) of ethylene signaling on electrolyte leakage (EL) and malondialdehyde (MDA) content. Under cold stress conditions, ACC-treated plant leaves had a greater level of EL and MDA than the untreated leaves. However, the EL and MDA values were lower in the Ag(+) regime versus the untreated. In addition, after 3 days of cold treatment, ACC remarkably reduced the content of soluble protein and also altered antioxidant enzyme activity. Under control (warm) condition, there was no significant effect of ACC on the performance of photosystem II (PS II) as monitored by chlorophyll α fluorescence transients. However, under cold stress, ACC inhibited the performance of PS II. Under cold condition, ACC remarkably reduced the performance index for energy conservation from excitation to the reduction of intersystem electron acceptors (PI(ABS)), the maximum quantum yield of primary photochemistry (φP0), the quantum yield of electron transport flux from Q(A) to Q(B) (φE0), and the efficiency/probability of electron transport (ΨE0). Simultaneously, ACC increased the values of specific energy fluxes for absorption (ABS/RC) and dissipation (DI0/RC) after 3 days of cold treatment. Additionally, under cold condition, exogenous ACC altered the expressions of several related genes implicated in the induction of cold tolerance (LEA, SOD, POD-1 and CBF1, EIN3-1, and EIN3-2). The present study thus suggests that ethylene affects the cold tolerance of Bermuda grass by impacting the antioxidant system, photosystem II, as well as the CBF transcriptional regulatory cascade.

Effects of cadmium exposure on growth and metabolic profile of bermudagrass [Cynodon dactylon (L.) Pers].

Metabolic responses to cadmium (Cd) may be associated with variations in Cd tolerance in plants. The objectives of this study were to examine changes in metabolic profiles in bermudagrass in response to Cd stress and to identify predominant metabolites associated with differential Cd tolerance using gas chromatography-mass spectrometry. Two genotypes of bermudagrass with contrasting Cd tolerance were exposed to 0 and 1.5 mM CdSO4 for 14 days in hydroponics. Physiological responses to Cd were evaluated by determining turf quality, growth rate, chlorophyll content and normalized relative transpiration. All these parameters exhibited higher tolerance in WB242 than in WB144. Cd treated WB144 transported more Cd to the shoot than in WB242. The metabolite analysis of leaf polar extracts revealed 39 Cd responsive metabolites in both genotypes, mainly consisting of amino acids, organic acids, sugars, fatty acids and others. A difference in the metabolic profiles was observed between the two bermudagrass genotypes exposed to Cd stress. Seven amino acids (norvaline, glycine, proline, serine, threonine, glutamic acid and gulonic acid), four organic acids (glyceric acid, oxoglutaric acid, citric acid and malic acid,) and three sugars (xylulose, galactose and talose) accumulated more in WB242 than WB144. However, compared to the control, WB144 accumulated higher quantities of sugars than WB242 in the Cd regime. The differential accumulation of these metabolites could be associated with the differential Cd tolerance in bermudagrass.

Identification of cadmium-resistant fungi related to Cd transportation in bermudagrass [Cynodon dactylon (L.) Pers].

Phytoremediation utilizing plants and microbes has been increasingly adopted as a green technology for cleaning up heavy metal polluted soils. Cd polluted soil and native bermudagrass from Liuyang and Zhuzhou in Hunan province of China were collected to investigate microbial diversity and isolate Cd resistant fungi, and then to determine the effect of Cd resistant fungi on Cd tolerance and transportation of bermudagrass. The functional diversity of microorganisms was evaluated using the BIOLOG Eco method. Cd-resistant fungi strain was isolated and identified as Aspergillus aculeatus based on the ribosomal internal transcribed spacer region sequence analysis. Bermudagrass was exposed to control, Cd only, and Cd plus A. aculeatus (Cd + A. aculeatus) with growth matrix (sawdust/sand = 3/1 in volume). Results indicated that Cd + A. aculeatus treated bermudagrass exhibited a higher photosynthetic activity compared to Cd only treated plants. Inoculation of A. aculeatus resulted in a decrease in stem and leaf Cd concentrations, to a greater extent for Cd-sensitive than for Cd-tolerant genotype. However, inoculation of A. aculeatus increased root Cd concentration under Cd stress conditions, significantly elevated soil pH, and decreased soil water-soluble Cd concentration. These results suggested that A. aculeatus might be potentially applied to improve Cd tolerance and to reduce Cd transportation to shoot of bermudagrass.

Classification of genetic variation for cadmium tolerance in Bermudagrass [Cynodon dactylon (L.) Pers.] using physiological traits and molecular markers.

Cadmium (Cd) is one of the most toxic pollutants that caused severe threats to animal and human health. Bermudagrass is a dominant species in Cd contaminated soils, which can prevent Cd flow and spread. The objectives of this study were to determine the genetic variations in major physiological traits related to Cd tolerance in six populations of Bermudagrass collected from China, and to examine the genetic diversity and relationships among these accessions that vary in Cd tolerance using molecular markers. Plants of 120 accessions (116 natural accessions and 4 commercial cultivars) were exposed to 0 (i.e. control) or 1.5 mM CdSO4·8/3H2O for 3 weeks in hydroponic culture. Turf quality, transpiration rate, chlorophyll content, leaf water content and growth rate showed wide phenotypic variation. The membership function method was used to comprehensively evaluate Cd-tolerance. According to the average subordinate function value, four accessions were classified as the most tolerant genotypes and four accessions as Cd-sensitive genotypes. The trend of Cd tolerance among the six studied populations was as follows: Hunan > South China > North China > Central China > West South China and Xinjiang population. Phylogenetic analysis revealed that the majority of accessions from the same or adjacent regions were clustered into the same groups or subgroups, and the accessions with similar cadmium tolerance displayed a close phylogenetic relationship. Screening genetically diverse germplasm by combining the physiological traits and molecular markers could prove useful in developing Cd-tolerant Bermudagrass for the remediation of mill tailings and heavy metal polluted soils.

Antioxidant responses and gene expression in perennial ryegrass (Lolium perenne L.) under cadmium stress.

Perennial ryegrass (Lolium perenne L.), widely used in temperate climates, is one of turf grasses that enrich cadmium (Cd). The objective of this study was to explore the physiological responses and candidate gene expression in perennial ryegrass to Cd stress. Grasses were subjected to three levels of 0, 0.2, and 0.5 mM Cd for 7 days in the greenhouse. The results indicated that soluble protein content was lower in the Cd-treated perennial ryegrass compared to untreated plants. The Cd-treated perennial ryegrass exhibited a greater level of malondialdehyde and activity of the peroxidase (POD), catalase, and superoxide dismutase (SOD) relative to the control. The Cd stress induced up-regulated expression of FeSOD, MnSOD, Chl Cu/ZnSOD, Cyt Cu/ZnSOD, APX, GPX, GR and POD at 4-24 h after treatment began for perennial ryegrass. Results suggested that the gene transcript profile was related to the enzyme activity under Cd stress.