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.

A comprehensive assessment of photosynthetic acclimation to shade in C4 grass (Cynodon dactylon (L.) Pers.).

BACKGROUND: Light deficit in shaded environment critically impacts the growth and development of turf plants. Despite this fact, past research has predominantly concentrated on shade avoidance rather than shade tolerance. To address this, our study examined the photosynthetic adjustments of Bermudagrass when exposed to varying intensities of shade to gain an integrative understanding of the shade response of C4 turfgrass. RESULTS: We observed alterations in photosynthetic pigment-proteins, electron transport and its associated carbon and nitrogen assimilation, along with ROS-scavenging enzyme activity in shaded conditions. Mild shade enriched Chl b and LHC transcripts, while severe shade promoted Chl a, carotenoids and photosynthetic electron transfer beyond QA- (ET0/RC, φE0, Ψ0). The study also highlighted differential effects of shade on leaf and root components. For example, Soluble sugar content varied between leaves and roots as shade diminished SPS, SUT1 but upregulated BAM. Furthermore, we observed that shading decreased the transcriptional level of genes involving in nitrogen assimilation (e.g. NR) and SOD, POD, CAT enzyme activities in leaves, even though it increased in roots. CONCLUSIONS: As shade intensity increased, considerable changes were noted in light energy conversion and photosynthetic metabolism processes along the electron transport chain axis. Our study thus provides valuable theoretical groundwork for understanding how C4 grass acclimates to shade tolerance.

Involvement of cell cycle and ion transferring in the salt stress responses of alfalfa varieties at different development stages.

BACKGROUND: Alfalfa (Medicago sativa) is the worldwide major feed crop for livestock. However, forage quality and productivity are reduced by salt stress, which is a common issue in alfalfa-growing regions. The relative salt tolerance is changed during plant life cycle. This research aimed to investigate the relative salt tolerance and the underlying mechanisms of two alfalfa varieties at different developmental stages. RESULTS: Two alfalfa varieties, "Zhongmu No.1 (ZM1)" and "D4V", with varying salt tolerance, were subjected to salt stress (0, 100, 150 mM NaCl). When the germinated seeds were exposed to salt stress, D4V exhibited enhanced primary root growth compared to ZM1 due to the maintenance of meristem size, sustained or increased expression of cell cycle-related genes, greater activity of antioxidant enzymes and higher level of IAA. These findings indicated that D4V was more tolerant than ZM1 at early developmental stage. However, when young seedlings were exposed to salt stress, ZM1 displayed a lighter wilted phenotype and leaf cell death, higher biomass and nutritional quality, lower relative electrolytic leakage (EL) and malondialdehyde (MDA) concentration. In addition, ZM1 obtained a greater antioxidant capacity in leaves, indicated by less accumulation of hydrogen peroxide (H2O2) and higher activity of antioxidant enzymes. Further ionic tissue-distribution analysis identified that ZM1 accumulated less Na+ and more K+ in leaves and stems, resulting in lower Na+/K+ ratio, because of possessing higher expression of ion transporters and sensitivity of stomata closure. Therefore, the relative salt tolerance of ZM1 and D4V was reversed at young seedling stages, with the young seedlings of the former being more salt-tolerant. CONCLUSION: Our data revealed the changes of relative order of salt tolerance between alfalfa varieties as they develop. Meristem activity in primary root tips and ion transferring at young seedling stages were underlying mechanisms that resulted in differences in salt tolerance at different developmental stages.

Comparative genomics reveals the molecular mechanism of salt adaptation for zoysiagrasses.

BACKGROUND: Zoysiagrass (Zoysia spp.) is a warm-season turfgrass. It is widely used as turfgrasses throughout the world, offers good turf qualities, including salt tolerance, resistance to drought and heat. However, the underlying genetic mechanism of zoysiagrass responsive to salt stress remains largely unexplored. RESULTS: In present study, we performed a whole-genome comparative analysis for ten plant genomes. Evolutionary analysis revealed that Chloridoideae diverged from Panicoideae approximately 33.7 million years ago (Mya), and the phylogenetic relationship among three zoysiagrasses species suggested that Zoysia matrella may represent an interspecific hybrid between Zoysia japonica and Zoysia pacifica. Genomic synteny indicated that Zoysia underwent a genus-specific whole-genome duplication (WGD) event approximately 20.8 Mya. The expression bais of homologous genes between the two subgenomes suggested that the B subgenome of Z. japonica contributes to salt tolerance. In additon, comparative genomic analyses revealed that the salt adaptation of Zoysia is likely attributable to the expanded cytochrome P450 and ABA biosynthetic gene families. Furthermore, we further found that many duplicated genes from the extra WGD event exhibited distinct functional divergence in response to salt stress using transcriptomic analysis, suggesting that this WGD event contributed to strong resistance to salt stress. CONCLUSIONS: Here, our results revealed that expanded cytochrome P450 and ABA biosynthetic gene families, and many of those duplicated genes from recent zoysia-specific WGD event contributed to salt adaptation of zoysiagrass, which provided insight into the genetic underpinning of salt adaptation and valuable information for further studies on salt stress-related traits in Zoysia.

Stress memory gene FaHSP17.8-CII controls thermotolerance via remodeling PSII and ROS signaling in tall fescue.

High temperature is the most limiting factor in the growth of cool-season turfgrass. To cope with high-temperature stress, grass often adopt a memory response by remembering one past recurring stress and preparing a quicker and more robust reaction to the next stress exposure. However, little is known about how stress memory genes regulate the thermomemory response in cool-season turfgrass. Here, we characterized a transcriptional memory gene, Fa-heat shock protein 17.8 Class II (FaHSP17.8-CII) in a cool-season turfgrass species, tall fescue (Festuca arundinacea Schreb.). The thermomemory of FaHSP17.8-CII continued for more than 4 d and was associated with a high H3K4me3 level in tall fescue under heat stress (HS). Furthermore, heat acclimation or priming (ACC)-induced reactive oxygen species (ROS) accumulation and photosystem II (PSII) electron transport were memorable, and this memory response was controlled by FaHSP17.8-CII. In the fahsp17.8-CII mutant generated using CRISPR/Cas9, ACC+HS did not substantially block the ROS accumulation, the degeneration of chloroplast ultra-structure, and the inhibition of PSII activity compared with HS alone. However, overexpression of FaHSP17.8-CII in tall fescue reduced ROS accumulation and chloroplast ultra-structure damage, and improved chlorophyll content and PSII activity under ACC+HS compared with that HS alone. These findings unveil a FaHSP17.8-CII-PSII-ROS module regulating transcriptional memory to enhance thermotolerance in cool-season turfgrass.

Genetic manipulation of bermudagrass photosynthetic biosynthesis using Agrobacterium-mediated transformation.

Bermudagrass is one of the most extensively used warm-season grasses. It is widely used in landscaping, stadium construction and soil remediation due to its excellent regeneration, trampling and stress tolerances. However, studies on its regulatory mechanism and variety improvement by genetic engineering are still at a standstill, owing to its genetic variability and intrinsic limits linked with some resistance to Agrobacterium infection. In this study, we established a higher efficient Agrobacterium-mediated transformation via screening for vital embryogenic callus and improving infection efficiency. The superior callus was light yellow, hard granular and compact, determined with a differentiation rate of more than 95%. The optimized infestation courses by gentle shaking, vacuuming and sonicating were used. The infested calluses were co-cultured for 3 days, followed by desiccation treatments for 1 day to get higher infection efficiency. Then the CdHEMA1 gene, essential for chlorophyll biosynthesis, was cloned and transferred into bermudagrass to validate the aforementioned optimization procedures integrally. Molecular-level analyses indicated that the CdHEMA1 gene had successfully integrated and was greatly increased in transgenic seedlings. Results of the photosynthetic capacity assessment showed that CdHEMA1 overexpression may considerably enhance the contents of photosynthetic pigments, OJIP curve and reaction center density (RC/CSo) to absorb (ABS/CSo, ABS/CSM) and capture (TRo/CSo) more light energy, hence improve the performance indices PIABS and PICS compared to the wild type. The successful completion of this project would provide a solid platform for further gene function study and molecular breeding of bermudagrass.

Transcriptome analysis of perennial ryegrass reveals the regulatory role of Aspergillus aculeatus under salt stress.

Perennial ryegrass (Lolium perenne) is an important turf grass and forage grass with moderately tolerant to salinity stress. Aspergillus aculeatus has been documented to involved in salt stress response of perennial ryegrass, while the A. aculeatus-mediated molecular mechanisms are unclear. Therefore, to investigate the molecular mechanisms underlying A. aculeatus-mediated salt tolerance, the comprehensive transcriptome analysis of the perennial ryegrass roots was performed. Twelve cDNA libraries from roots were constructed after 12 h of plant-fungus cocultivation under 300 mM salt stress concentrations. A total of 21,915 differentially expressed genes (DEGs) were identified through pairwise comparisons. Enrichment analysis revealed that potentially important A. aculeatus-induced salt responsive genes belonging to specific categories, such as hormonal metabolism (auxin and salicylic acid metabolism related genes), secondary metabolism (flavonoid's metabolism related genes) and transcription factors (MYB, HSF and AP2/EREBP family). In addition, weighted gene co-expression network analysis (WGCNA) showed that blue and black modules were significantly positively correlated with the peroxidase activity and proline content, then the hub genes within these two modules were further identified. Taken together, we found the categories of A. aculeatus-induced salt responsive genes, revealing underlying fungus-induced molecular mechanisms of salt stress response in perennial ryegrass roots. Besides, fungus-induced salt-tolerant hub genes represent a foundation for further exploring the molecular mechanisms.

Physiological integration between Bermudagrass ramets improves overall salt resistance under heterogeneous salt stress.

Connected ramets of colonal plants often suffer from different environmental conditions such as light, nutrient, and stress. Colonal Bermudagrass (Cynodon dactylon [L.] Pers.) can form interconnected ramets and this connection facilitates the tolerance to abiotic stress, which is a kind of physiological integration. However, how bermudagrass responds to heterogeneously distributed salt stress needs to be further elucidated. Here, we demonstrated that severance of stolons aggravated the damage of salt-stressed ramets, displaying higher relative electrolytic leakage (EL), lower content of chlorophyll, higher accumulation of Na+ , and serious oxidative damages. This finding implied the positive effects of the physiological integration of bermudagrass on salt tolerance. The unstressed ramets connected with the stressed one were mildly injured, implying the supporting and sacrifice function of the unstressed ramets. Physiological integration did not mediate the translocation of Na+ among ramets, but induced a higher expression of salt overly sensitive (SOS) genes in the stressed ramets, consequently reducing the accumulation of Na+ in leaves and roots. In addition, physiological integration upregulated the genes expression and enzymes activity of catalase (CAT) and peroxidase (POD) in both stressed and unstressed ramets. This granted a stronger antioxidant ability of the whole clonal plants under salt stress. Enhanced Na+ transfer and increased reactive oxygen species (ROS) scavenging are mechanisms that likely contribute to the physiological integration leading to the salt tolerance of bermudagrass.

Aspergillus aculeatus enhances potassium uptake and photosynthetic characteristics in perennial ryegrass by increasing potassium availability.

AIM: Potassium (K) is a key determinant for plant development and productivity. However, more than 90% of K in the soil exists in an insoluble form. K-solubilizing microbes play an important role in the transformation of insoluble K. Thus, the objective of this study was to evaluate K-dissolving ability of Aspergillus aculeatus (F) and growth-promoting properties in perennial ryegrass. METHODS AND RESULTS: Perennial ryegrass inoculated with A. aculeatus exhibited enhanced soluble K accompanied with higher growth rate and turf quality, compared with the noninoculated regimen. In addition, A. aculeatus also played a primary role in increasing chlorophyll content and photosynthetic capacity of the plant exposed to LK+F (K-feldspar plus A. aculeatus) treatment, compared with the CK (control, no K-feldspar and A. aculeatus), F (only A. aculeatus) and LK (only K-feldspar) groups. Furthermore, the antioxidase activities (CAT and POD) were significantly increased while the oxidative damage (EL and MDA) was dramatically decreased in the LK+F group compared to the LK (K-feldspar) group. Finally, in perennial ryegrass leaves, the genes expression levels of HAK8, HAK12 and HKT18 were obviously elevated in the LK+F group, compared to the CK, F and LK groups. CONCLUSION: We concluded that A. aculeatus could solubilize K from bound form and be considered as K-solubilizing biofertilizer through supplementing K in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillusaculeatus has the potential to be used as a biofertilizer in sustainable agriculture.

Comprehensive transcriptional analysis reveals salt stress-regulated key pathways, hub genes and time-specific responsive gene categories in common bermudagrass (Cynodon dactylon (L.) Pers.) roots.

BACKGROUND: Despite its good salt-tolerance level, key genes and pathways involved with temporal salt response of common bermudagrass (Cynodon dactylon (L.) Pers.) have not been explored. Therefore, in this study, to understand the underlying regulatory mechanism following the different period of salt exposure, a comprehensive transcriptome analysis of the bermudagrass roots was conducted. RESULTS: The transcripts regulated after 1 h, 6 h, or 24 h of hydroponic exposure to 200 mM NaCl in the roots of bermudagrass were investigated. Dataset series analysis revealed 16 distinct temporal salt-responsive expression profiles. Enrichment analysis identified potentially important salt responsive genes belonging to specific categories, such as hormonal metabolism, secondary metabolism, misc., cell wall, transcription factors and genes encoded a series of transporters. Weighted gene co-expression network analysis (WGCNA) revealed that lavenderblush2 and brown4 modules were significantly positively correlated with the proline content and peroxidase activity and hub genes within these two modules were further determined. Besides, after 1 h of salt treatment, genes belonging to categories such as signalling receptor kinase, transcription factors, tetrapyrrole synthesis and lipid metabolism were immediately and exclusively up-enriched compared to the subsequent time points, which indicated fast-acting and immediate physiological responses. Genes involved in secondary metabolite biosynthesis such as simple phenols, glucosinolates, isoflavones and tocopherol biosynthesis were exclusively up-regulated after 24 h of salt treatment, suggesting a slightly slower reaction of metabolic adjustment. CONCLUSION: Here, we revealed salt-responsive genes belonging to categories that were commonly or differentially expressed in short-term salt stress, suggesting possible adaptive salt response mechanisms in roots. Also, the distinctive salt-response pathways and potential salt-tolerant hub genes investigated can provide useful future references to explore the molecular mechanisms of bermudagrass.

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.

High temperature damage to fatty acids and carbohydrate metabolism in tall fescue by coupling deep transcriptome and metabolome analysis.

High temperature damage impairs the growth of tall fescue by inhibiting secondary metabolites. Little is known about the regulation pattern of the fatty acids and carbohydrate metabolism at the whole-transcriptome level in tall fescue under high temperature stress. Here, two tall fescue genotypes, heat tolerant PI578718 and heat sensitive PI234881 were subjected to high temperature stress for 36 h. PI 578718 showed higher SPAD chloroplast value, lower EL and leaf injury than PI 234881 during the first 36 h high-temperature stress. Furthermore, by transcriptomic analysis, 121 genes were found to be induced during the second energy production phase in tall fescue exposed to high-temperature conditions, indicating that there may be one energy-sensing system in cool-season turfgrass to adapt high-temperature conditions. PI 578718 showed higher differentially expressed unigenes involved in fatty acids and carbohydrate metabolism compared with PI 234881 for 36 h heat stress. Interestingly, a metabolomic analysis using GC-MS uncovered that the sugars and sugar alcohol accounted for more than 65.06% of the total 41 metabolites content and high-temperature elevated the rate to 82.89-91.16% in PI 578718. High-temperature damage decreased the rate of fatty acid in the total 41 metabolites content and PI 578718 showed lower content than in PI 234881, which might be attributed to the down-regulated genes in fatty acid biosynthesis pathway in tall fescue. The integration of deep transcriptome and metabolome analyses provides systems-wide datasets to facilitate the identification of crucial regulation factors in cool-season turfgrass in response to high-temperature damage.

Characterization of the Cd-resistant fungus Aspergillus aculeatus and its potential for increasing the antioxidant activity and photosynthetic efficiency of rice.

Considerable evidence exists that microorganisms play a significant role in the remediation of soil contaminated with heavy metals. Aspergillus aculeatus (A. aculeatus) isolated from Cd-polluted soil has been shown to increase the tolerance of turfgrasses to Cd stress. In this study, we assessed the tolerance, biosorption capacity for Cd and surface characteristics of this fungus and investigated the effect of plant inoculation with A. aculeatus on the lipid peroxidation, antioxidant activities and photosynthetic rates in rice cultivated in Cd-contaminated soil. The results indicated that the removal efficiency of A. aculeatus was 46.8% at a Cd concentration of 10 mg L-1. The A. aculeatus strains had the capacity to produce indole acetic acid, siderophore, and 1-aminocyclopropane-1-carboxylate deaminase and to solubilize phosphate. The O2- accumulation and the amount of MDA in rice roots inoculated with A. aculeatus were significantly lower than those in uninoculated plants. Nevertheless, no decrease in leaf ROS accumulation and photosynthetic activity was observed between the inoculated and uninoculated plants. Inoculation with A. aculeatus contained more of the ROS-scavenging metabolite GSH, a higher GSH/GSSG ratio, and higher antioxidative enzyme (SOD, POD, and CAT) activities, possibly explaining the lower ROS concentrations observed in inoculated roots in the presence of Cd. These results suggest that application of A. aculeatus has the potential to protect crops against Cd stress.

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.

Transcriptome profilings of two tall fescue (Festuca arundinacea) cultivars in response to lead (Pb) stress.

BACKGROUND: Lead (Pb) is one of the most toxic heavy metal environmental pollutants. Tall fescue is an important cold season turf grass which can tolerate and accumulate substantial amount of Pb. To estimate genes related to Pb response and the molecular mechanism associated with Pb tolerance and accumulation, we analyzed the transcriptome of tall fescue in response to Pb treatment. RESULTS: RNA-sequencing was performed in two tall fescue cultivars, Pb tolerant Silverado and Pb sensitive AST7001. A total of 810,146 assembled unique transcripts representing 25,415 unigenes were obtained from the tall fescue leaves. Among the panel, 3,696 differentially expressed genes (DEGs) were detected between the Pb treated (1000 mg/L) and untreated samples. Gene ontology (GO) and pathway enrichment analysis demonstrated that the DEGs were mainly implicated in energy metabolism, metabolism of terpenoids and polyketides, and carbohydrate metabolism related pathways. The expression patterns of 16 randomly selected genes were in consistent with that from the Solexa analysis using quantitative reverse-transcription PCR. In addition, compared to the common transcriptional response to Pb stress in both cultivars, the regulation of numerous genes including those involved in zeatin biosynthesis, limonene and pinene degradation, phagosome was exclusive to one cultivar. CONCLUSIONS: The tall fescue assembled transcriptome provided substantial molecular resources for further genomics analysis of turfgrass in response to heavy metal stress. The significant expression difference of specific unigenes may account for Pb tolerance or accumulation in two different tall fescue cultivars. This study provided new insights for the investigation of the molecular basis of Pb tolerance and accumulation in tall fescue as well as other related turf grass species.

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.

Stress memory induced transcriptional and metabolic changes of perennial ryegrass (Lolium perenne) in response to salt stress.

Preexposure to a stress could induce stable signals and reactions on plant physiology and gene expression during future encounters as a 'stress memory'. In this study, we found that two trainable genes, BPSP encoding putative brown plant hopper susceptibility protein and sucs encoding sucrose synthase displayed transcriptional memory for their considerably higher transcript levels during two or more subsequent stresses (S3, S4) relative to the initial stress (S0), and their expression returning to basal transcript levels (non-stressed) during the recovery states (R1, R2 and R3). Removing the repetitive stress/recovery exercise, activated transcriptional memory from two trainable genes persisted for at least 4 days in perennial ryegrass. The pretrainable genes with stress memory effort had higher response to the subsequent elevated NaCl concentration treatment than the non-trainable plants, which was confirmed by lower electrolyte leakage and minimum H2 O2 and O2 (-) accumulation. Salt stress elevated the content of 41 metabolites in perennial ryegrass leaves, and sugars and sugar alcohol accounted for more than 74.1% of the total metabolite content. The salt stress memory was associated with higher contents of 11 sugars and 1 sugar alcohol in the pretrainable grass leaves. Similarly, six sugars showed greater content in the pretrainable grass roots. These novel phenomena associated with transcriptional memory and metabolite profiles could lead to new insights into improving plant salinity acclimation process.

Photosynthesis, antioxidant system and gene expression of bermudagrass in response to low temperature and salt stress.

There is widespread distribution of salinized lands in northern China. Harnessing such land is essential to environmental health. Bermudagrass [Cynodon dactylon (L.) Pers.] has the potential to improve the salinized lands. However, low temperature remarkably limits the growth of bermudagrass in winter. Currently, there is no information about the interaction of cold and salt in this plant. Hence, the objectives of this study were to figure out the effects of combined cold and salinity stress on bermudagrass. In this study, 4 °C and 200 mM salt solution was used as cold and salt treatments respectively while 4 °C along with 200 mM salt solution were applied as combined stress. After 5 days treatment, bermudagrass displayed a dramatic decline in the turf quality and chlorophyll content, but higher malonaldehyde, electrolyte leakage, hydrogen peroxide content, antioxidant enzyme activity in the combined stress regime as compared to cold or salt treated alone. Analysis of chlorophyll a revealed that the combined stress aggravated stress-induced inhibition of photosystem II. In addition, the expressions of stress-related genes were up-regulated with a lower expression level when cold and salt applied together. In summary, the grass exposed to combined stress presented a relatively lower stress tolerance and suffered a more severe damage than grass grown in the other regimes. These findings are crucial for elucidating the molecular mechanisms of cold and salt combined stress in bermudagrass, and provide information for breeding programs to select and develop bermudagrass cultivars that are suitable for improvement of the northern China salinized land.

RNA-seq for gene identification and transcript profiling in relation to root growth of bermudagrass (Cynodon dactylon) under salinity stress.

BACKGROUND: Soil salinity is one of the most significant abiotic stresses affecting plant shoots and roots growth. The adjustment of root architecture to spatio-temporal heterogeneity in salinity is particularly critical for plant growth and survival. Bermudagrass (Cynodon dactylon) is a widely used turf and forage perennial grass with a high degree of salinity tolerance. Salinity appears to stimulate the growth of roots and decrease their mortality in tolerant bermudagrass. To estimate a broad spectrum of genes related to root elongation affected by salt stress and the molecular mechanisms that control the positive response of root architecture to salinity, we analyzed the transcriptome of bermudagrass root tips in response to salinity. RESULTS: RNA-sequencing was performed in root tips of two bermudagrass genotypes contrasting in salt tolerance. A total of 237,850,130 high quality clean reads were generated and 250,359 transcripts were assembled with an average length of 1115 bp. Totally, 103,324 unigenes obtained with 53,765 unigenes (52 %) successfully annotated in databases. Bioinformatics analysis indicated that major transcription factor (TF) families linked to stress responses and growth regulation (MYB, bHLH, WRKY) were differentially expressed in root tips of bermudagrass under salinity. In addition, genes related to cell wall loosening and stiffening (xyloglucan endotransglucosylase/hydrolases, peroxidases) were identified. CONCLUSIONS: RNA-seq analysis identified candidate genes encoding TFs involved in the regulation of lignin synthesis, reactive oxygen species (ROS) homeostasis controlled by peroxidases, and the regulation of phytohormone signaling that promote cell wall loosening and therefore root growth under salinity.