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.

Root system architecture and genomic plasticity to salinity provide insights into salt-tolerant traits in tall fescue.

Salinity is detrimental to soil health, plant growth, and crop productivity. Understanding salt tolerance mechanisms offers the potential to introduce superior crops, especially in coastal regions. Root system architecture (RSA) plasticity is vital for plant salt stress adaptation. Tall fescue is a promising forage grass in saline regions with scarce RSA studies. Here, we used the computer-integrated and -automated programs EZ-Rhizo II and ROOT-Vis II to analyze and identify natural RSA variations and adaptability to high salt stress at physiological and genetic levels in 17 global tall fescue accessions. Total root length rather than the number of lateral roots contribute more to water uptake and could be used to separate salt-tolerant (LS-11) and -sensitive accessions (PI531230). Comparative evaluation of LS-11 and PI531230 demonstrated that the lateral root length rather than the main root contributed more towards the total root length in LS-11. Also, high water uptake was associated with a larger lateral root vector and position while low water intake was associated with an insignificant correlation between root length, vector, and position. To examine candidate gene expression, we performed transcriptome and transcription analyses using high-throughput RNA sequencing and real-time quantitative PCR, respectively of the lateral and main roots. The main root displayed more differentially expressed genes than the lateral root. A Poisson comparison of LS-11 vs PI531230 demonstrated significant upregulation of PLASMA MEMBRANE AQUAPORIN 1 and AUXIN RESPONSE FACTOR 22 in both the main and lateral root, which are associated with transmembrane water transport and the auxin-activated signaling system, respectively. There is also an upregulation of BASIC HELIX-LOOP-HELIX 5 in the main root and a downregulation in the lateral root, which is ascribed to sodium ion transmembrane transport, as well as an upregulation of THE MEDIATOR COMPLEX 1 assigned to water transport in the lateral root and a downregulation in the main root. Gene-protein interaction analysis found that more genes interacting with aquaporins proteins were upregulated in the lateral root than in the main root. We inferred that deeper main roots with longer lateral roots emanating from the bottom of the main root were ideal for tall fescue water uptake and salt tolerance, rather than many shallow roots, and that, while both main lateral roots may play similar roles in salt sensing and water uptake, there are intrinsic genomic differences.

CdWRKY2 transcription factor modulates salt oversensitivity in bermudagrass [Cynodon dactylon (L.) Pers.].

Common bermudagrass [Cynodon dactylon (L.) Pers.] has higher utilization potential on saline soil due to its high yield potential and excellent stress tolerance. However, key functional genes have not been well studied partly due to its hard transformation. Here, bermudagrass "Wrangler" successfully overexpressing CdWRKY2 exhibited significantly enhanced salt and ABA sensitivity with severe inhibition of shoot and root growth compared to the transgenic negative line. The reduced auxin accumulation and higher ABA sensitivity of the lateral roots (LR) under salt stress were observed in CdWRKY2 overexpression Arabidopsis lines. IAA application could rescue or partially rescue the salt hypersensitivity of root growth inhibition in CdWRKY2-overexpressing Arabidopsis and bermudagrass, respectively. Subsequent experiments in Arabidopsis indicated that CdWRKY2 could directly bind to the promoter region of AtWRKY46 and downregulated its expression to further upregulate the expression of ABA and auxin pathway-related genes. Moreover, CdWRKY2 overexpression in mapk3 background Arabidopsis could partly rescue the salt-inhibited LR growth caused by CdWRKY2 overexpression. These results indicated that CdWRKY2 could negatively regulate LR growth under salt stress via the regulation of ABA signaling and auxin homeostasis, which partly rely on AtMAPK3 function. CdWRKY2 and its homologue genes could also be useful targets for genetic engineering of salinity-tolerance plants.

Irrigated barley-grass pea crop mixtures can revive soil microbial activities and alleviate salinity in desertic conditions of southern Morocco.

Soil salinity adversely limits crop and soil health, and this can be reversed by cropping systems where species exclude salts and activate microbial nutrient cycling. A randomized complete block design experiment was established in Laayoune-Morocco to evaluate the influence of irrigated grass pea and barley monocrops or combined together in 50-50% and 70-30% mixtures against soil salinity and CO2-C flux in sites with varying salinity. Site by treatment interaction significantly influenced (p < 0.05) soil salinity and CO2-C flux. Salinity reduced by 37 to 68 dS m-1 in highly saline soils across season regardless of treatment and barley monocrop retained the least salinity (15 dS m-1). Same applied to sites with low (1 to 2 dS m-1) and medium (2 to 5 dS m-1) salinity although less pronounced. The 70-30% grass pea, barley mixture maintained the greatest CO2-C flux in soils with low salinity and marginally enhancing soil active carbon (130 to 229 mg kg-1 soil) in different sites. Increasingly saline water filled pore space devastated CO2-C flux, although this process recovered under barley at extreme salinity. Overall, barley in mixture with grass pea can alleviate salinity and accelerate microbial carbon sequestration if irrigation is modulated in shallow desertic soils.

Diversity analysis of agronomic and nutritional traits of hybrid offspring of forage bermudagrass.

Because of its excellent stress resistance and forage quality, the forage bermudagrass hybrid population had attracted the attention of scientific researchers in recent years. Studying its diversity could promote the breeding of desirable varieties. The variability in agronomic traits including fresh weight, dry weight, ash content, crude protein content, crude fat, phosphorus content, and relative feed value for 56 bermudagrass was investigated using Wrangler as an experimental reference. Grey correlation analysis and cluster analysis were employed to screen bermudagrass with high yield and superior quality. WCF-34 had the highest 2-year fresh weight (109,773.3 kg/ha), WCF-37 had the highest 2-year dry weight (31,951.6 kg/ha), WCF-24 had the lowest Ash content (7.46%), WCF-26 had the highest crude protein content (16.27%), WCF-27 had the highest curde fat content (3.58%), WCF-13 had the highest P content (0.45%), and WCF-42 had the highest relative feed value (95.32). Combining the results of grey relational analysis and cluster analysis, WCF-42, WCF-34, WCF-38, WCF-37, and WCF-40 were selected as high-quality bermudagrass. Through comprehensive analysis of the agronomic characters of bermudagrass, five bermudagrass were selected, the outcomes of this study would provide a theoretical basis for the breeding and genetic enhancement of bermudagrass.

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.

Influence of cutting time interval and season on productivity, nutrient partitioning, and forage quality of blue panicgrass (Panicum antidotale Retz.) under saline irrigation in Southern region of Morocco.

Salinity has become a major issue in various parts of the world negatively impacting agricultural activities and leading to diminished crop potential and lower yields. Such situation calls for urgent interventions such as adopting salt-tolerant crops to fill the gap in food and feed availability. Blue panicgrass (Panicum antidotale Retz.) is a promising salt-tolerant forage crop that has shown an appropriate adaptation and performance in the saline, arid, and desertic environments of southern Morocco. However, for obtaining a highest forage productivity with nutritional quality, optimization of the cutting interval is required. Thus, the objective of this study was to determine the optimal cutting time interval allowing high forage production and quality under high salinity conditions. This experiment was conducted over one entire year covering the summer and winter seasons. The effect of five cutting time intervals on selected agro-morphological traits, crop productivity, mineral nutrient accumulation, and forage quality of blue panicgrass in the region of Laayoune, southern Morocco. The finding of this study recommend that cutting blue panicgrass every 40 days maximized the annual fresh and dry forage yield as well as the protein yield, which reached 74, 22, and 2.9 t/ha, respectively. This study also revealed a significant effect of the season on both productivity and quality. However, forage yield declined during the winter and increased during the summer, while protein content increased during winter compared to summer. The mineral nutrient partitioning between shoots and roots, especially the K+/Na+ ratio, indicated that blue panicgrass has salt tolerance mechanism as it excluded sodium from the roots and compartmentalized it in the leaves. In conclusion, there is a potential of blue panicgrass on sustaining forage production under salt-affected drylands, as demonstrated by the response to two key questions: (a) a technical question to farmers for its adoption such as at which interval should blue panicgrass be harvested maximizing both forage yield and quality? And (b) a scientific question on how does blue panicgrass maintain high K+/Na+ ratio to cope with salinity stress?

Salt-responsive bermudagrass microRNAs and insights into light reaction photosynthetic performance.

Introduction: Bermudagrass (Cynodon dactylon L.) is a warm-season grass with high drought and salt tolerance. However, its cultivation as a silage crop is limited by its lower forage value when compared to other C4 crops. Because of its high genetic variability in abiotic stress tolerance, bermudagrass-mediated genetic breeding offers significant promise for introducing alternative fodder crops in saline and drought-affected regions, and improved photosynthetic capacity is one way for increasing forage yield. Methods: Here, we used RNA sequencing to profile miRNAs in two bermudagrass genotypes with contrasting salt tolerance growing under saline conditions. Results: Putatively, 536 miRNA variants were salt-inducible, with the majority being downregulated in salt-tolerant vs sensitive varieties. Also, seven miRNAs putatively targeted 6 genes which were significantly annotated to light reaction photosynthesis. Among the microRNAs, highly abundant miRNA171f in the salt tolerant regime targeted Pentatricopeptide repeat-containing protein and dehydrogenase family 3 member F1 both annotated to electron transport and Light harvesting protein complex 1 genes annotated to light photosynthetic reaction in salt tolerant regime vs salt sensitive counterparts. To facilitate genetic breeding for photosynthetic capacity, we overexpressed miR171f in Medicago tracantula which resulted in a substantial increase in the chlorophyll transient curve, electron transport rate, quantum yield of photosystem II non photochemical quenching, NADPH and biomass accumulation under saline conditions while its targets were downregulated. At ambient light level the electron transport was negatively correlated with all parameters while the NADPH was positively associated higher dry matter in mutants. Discussion: These results demonstrate that miR171f improves photosynthetic performance and dry matter accumulation via transcriptional repression of genes in the electron transport pathway under saline conditions and thus a target for breeding.

The extended day length promotes earlier flowering of bermudagrass.

Day length is a very critical environmental factor affecting plant growth and development. The extension of light application time has been shown to promote flowering in the long-day plant and to shorten breeding time in some crops. However, previous research on the regulation of bermudagrass flowering by light application time is scarce. Therefore, this study investigated the effect of day length on the growth and flowering of bermudagrass by prolonging the light application time in a controlled greenhouse. Three different light application times were set up in the experiment: 22/2 h (22 hours light/2 hours dark), 18/6 h (18 hours light/6 hours dark), 14/10 h (14 hours light/10 hours dark). Results showed that extending the light application time not only promoted the growth of bermudagrass (plant height, fresh weight, dry weight) but also its nutrient uptake (nitrogen (N) and phosphorous (P) content). In addition, daily light integrals were different when flowering under different light application times. Most importantly, under the 22/2 h condition, flowering time was successfully reduced to 44 days for common bermudagrass (Cynodon dactylon [L.] pers) genotype A12359 and 36 days for African bermudagrass (Cynodon transvaalensis Burtt-Davy) genotype ABD11. This study demonstrated a successful method of bermudagrass flowering earlier than usual time by manipulating light application time which may provide useful insights for bermudagrass breeding.

Mitigation of salt stress on low temperature in bermudagrass: resistance and forage quality.

Climate change causes plants encountering several abiotic stresses simultaneously. Responses of plants to a single stress has been comprehensively studied, but it is hard to speculated infer the effects of stress combination based on these researches. Here, the response mechanism of bermudagrass to low temperature and salt treatment was investigated in this study. The results showed that low temperature (LT) treatment decreased the relative growth rate, chlorophyll fluorescence transient curve, biomass, and crude fat content of bermudagrass, whereas low temperature + salt (LT+S) treatment greatly undermined these declines. Furthermore, at 6 h and 17 d, the expression levels of glyoxalase I (GLYI), Cu-Zn/superoxide dismutase (Cu-Zn/SOD), peroxidase 2 (POD2), and oxidative enzyme 1(CAT1) in roots were considerably higher in the low temperature + salt treatment than in the low temperature treatment. Low temperature stress is more detrimental to bermudagrass, but mild salt addition can mitigate the damage by enhancing photosynthesis and improving the expression of antioxidant system genes (Cu-Zn/SOD, POD2 and CAT1) and glyoxalase system GLYI gene in roots. This study summarized the probable interaction mechanism of low temperature and salt stress on bermudagrass, which can provide beneficial reference for the growth of fodder in cold regions.

Crop Species Mechanisms and Ecosystem Services for Sustainable Forage Cropping Systems in Salt-Affected Arid Regions.

Soil salinity limits crop productivity in arid regions and it can be alleviated by crop synergies. A multivariate analysis of published data (n = 78) from arid and semiarid habitats across continents was conducted to determine the crop species mechanisms of salinity tolerance and synergies relevant for designing adapted forage cropping systems. Halophyte [Cynodon plectostachus (K. Schum.) Pilg.] and non-halophyte grasses (Lolium perenne L. and Panicum maximum Jacq.) clustered along increasing soil salinity. Halophytic grasses [Panicum antidotale Retz. and Dicanthum annulatum (Forssk.) Stapf] congregated with Medicago sativa L., a non-halophytic legume along a gradient of increasing photosynthesis. Halophytic grasses [Sporobolus spicatus (Vahl) Kunth, and Cynodon plectostachyus (K. Schum.) Pilg.] had strong yield-salinity correlations. Medicago sativa L. and Leptochloa fusca L. Kunth were ubiquitous in their forage biomass production along a continuum of medium to high salinity. Forage crude protein was strongly correlated with increasing salinity in halophytic grasses and non-halophytic legumes. Halophytes were identified with mechanisms to neutralize the soil sodium accumulation and forage productivity along an increasing salinity. Overall, halophytes-non-halophytes, grass-forbs, annual-perennials, and plant-bacteria-fungi synergies were identified which can potentially form cropping systems that can ameliorate saline soils and sustain forage productivity in salt-affected arid regions.

Moderately Reducing Nitrogen Application Ameliorates Salt-Induced Growth and Physiological Damage on Forage Bermudagrass.

Nitrogen (N) application is one of the most effective methods to alleviate salt-induced damage on plants. Forage bermudagrass has higher utilization potential on saline soil, but whether its N requirement changed under high salt stress has not been studied. Through examining plant growth-related traits, salt-stress-responsive physiological traits, photosynthesis, N metabolism, and forage quality supplied with different N concentrations under high salt stress (200 mM NaCl), we noticed that the optimum N requirement of forage bermudagrass reduced. When supplied with 10 mM N under higher salt stress, plants had a similar biomass, turf color, and chlorophyll content with plants supplied with 15 mM N, accompanied by a lower firing rate and Na+ content of leaves. The N content, crude protein, crude fat content, the expression of AMTs (ammonium transporters), NR (nitrate reductase), GS (glutamine synthetase), and GOGAT (glutamate synthetase), the chlorophyll fluorescence curve, and parameters of leaves (e.g., PIABS; PICS; ABS/RC; TRo/RC; ETo/RC) all peaked under 10 mM N under high salt stress instead of 15 mM N. Through exploring the proper N application under higher salt stress and its alleviation mechanisms, our results indicated that moderate reduction in N application under high salt level had a maximum promotion effect on the salt tolerance of forage bermudagrass without growth or forage quality inhibition. These response mechanisms obtained can provide a useful reference for N application in moderation rather than in excess on forage bermudagrass, especially in higher salinity areas.

Exploring the correlation between salt tolerance and yield: research advances and perspectives for salt-tolerant forage sorghum selection and genetic improvement.

MAIN CONCLUSION: Some salt stress response mechanisms can translate into sorghum forage yield and thus act as targets for genetic improvement. Sorghum is a drought-tolerant cereal that is widely grown in the vast Africa's arid and semi-arid areas. Apart from drought, salinity is a major abiotic factor that, in addition to natural causes, has been exacerbated by increased poor anthropological activities. The importance of sorghum as a forage crop in saline areas has yet to be fully realized. Despite intraspecific variation in salt tolerance, sorghum is generally moderately salt-tolerant, and its productivity in saline soils can be remarkably limited. This is due to the difficulty of replicating optimal field saline conditions due to the great heterogeneity of salt distribution in the soil. As a promising fodder crop for saline areas, classic phenotype-based selection methods can be integrated with modern -omics in breeding programs to simultaneously address salt tolerance and production. To enable future manipulation, selection, and genetic improvement of sorghum with high yield and salt tolerance, here, we explore the potential positive correlations between the reliable indices of sorghum performance under salt stress at the phenotypic and genotypic level. We then explore the potential role of modern selection and genetic improvement programs in incorporating these linked salt tolerance and yield traits and propose a mechanism for future studies.

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.

Exogenous Aspergillus aculeatus Enhances Drought and Heat Tolerance of Perennial Ryegrass.

Perennial ryegrass (Lolium perenne) is a cool-season grass whose growth and development are limited by drought and high temperature. Aspergillus aculeatus has been reported to promote plant growth and counteract the adverse effects of abiotic stresses. The objective of this study was to assess A. aculeatus-induced response mechanisms to drought and heat resistance in perennial ryegrass. We evaluated the physiological and biochemical markers of drought and heat stress based on the hormone homeostasis, photosynthesis, antioxidant enzymes activity, lipid peroxidation, and genes expression level. We found out that under drought and heat stress, A. aculeatus-inoculated leaves exhibited higher abscisic acid (ABA) and lower salicylic acid (SA) contents than non-inoculated regimes. In addition, under drought and heat stress, the fungus enhanced the photosynthetic performance, decreased the antioxidase activities, and mitigated membrane lipid peroxidation compared to non-inoculated regime. Furthermore, under drought stress, A. aculeatus induced a dramatic upregulation of sHSP17.8 and DREB1A and a downregulation of POD47, Cu/ZnSOD, and FeSOD genes. In addition, under heat stress, A. aculeatus-inoculated plants exhibited a higher expression level of HSP26.7a, sHSP17.8, and DREB1A while a lower expression level of POD47 and FeSOD than non-inoculated ones. Our results provide an evidence of the protective role of A. aculeatus in perennial ryegrass response to drought and heat stresses.

Moderately low nitrogen application mitigate the negative effects of salt stress on annual ryegrass seedlings.

Appropriate application of nitrogen (N) can alleviate the salt stress-induced damage on plants. This study explores the changes of nitrogen requirement in feeding annual ryegrass seedlings under mild salt concentrations (50 mM, 100 mM) plus its underlying mitigation mechanism. Results showed that low salt concentration decreased N requirement as observed from the increment in plant height and biomass at a relative low N level (2.0 mM not 5.0 mM). Under salt treatment, especially at 50 mM NaCl, the OJIP (Chl a fluorescence induction transient) curve and a series of performance indexes (PIABS, RC/CS0, ET0/CS0, ϕE0, ϕ0) peaked whereas DI0/RC, Vj and M0 were the lowest under moderately low N level (2.0 mM). In addition, under salt stress, moderately low N application could maintain the expression of NR (nitrate reductase) and GS (glutamine synthetase) encoding genes at a relatively stable level but had no effect on the expression of detected NRT (nitrate transporter) gene. The seedlings cultured at 2.0 mM N also exhibited the highest activity of CAT and POD antioxidant enzymes and the lowest MDA content and EL under relative low level of salt treatment. These results indicated that mild salt treatment of annual ryegrass seedlings might reduce N requirement while moderately low N application could promote their growth via regulating photosynthesis, alleviating ROS-induced (reactive oxygen species) damage and maintenance of N metabolism. These results also can provide useful reference for nitrogen application in moderation rather than in excess on annual ryegrass in mild or medium salinity areas through understanding the underlying response mechanisms.

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.

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.

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.

Comparative physiological and metabolomic analyses reveal mechanisms of Aspergillus aculeatus-mediated abiotic stress tolerance in tall fescue.

Aspergillus aculeatus has been shown to stimulate plant growth, but its role in plants abiotic stress tolerance and the underlying mechanisms are not fully documented. In this study, we investigated the mechanisms of A.aculeatus-mediated drought, heat and salt stress tolerance in tall fescue. The results showed that A.aculeatus inoculation improved drought and heat stress tolerance in tall fescue as observed from its effect on turf quality (TQ) and leaf relative water content (LWC). In the same stress conditions, A.aculeatus alleviated reactive oxygen species (ROS) induced burst and cell damage, as indicated by lower H2O2, electrolyte leakage (EL) and malondialdehyde (MDA) levels. Additionally, the A.aculeatus inoculated plants exhibited higher photosynthetic efficiency than uninoculated plants under drought, heat and salt stress conditions. The fungus reduced the uptake of Na+, and inoculated plants showed lower Na+/K+, Na+/Ca2+and Na+/Mg2+ ratios compared to uninoculated ones under salt stress. Furthermore, comparative metabolomic analysis showed that A.aculeatus exclusively increased amino acid (such as proline and glycine) and sugar (such as glucose, fructose, sorbose, and talose) accumulation under drought and heat stress. However, there were no differences between inoculated and uninoculated plants except for changes in H2O2 level, Na+ in the root and photosynthetic efficiency under salt stress. Taken together, this study provides the first evidence of the protective roles of A.aculeatus in the tall fescue response to abiotic stresses, partially via protection of photosynthesis and modulation of metabolic homeostasis.