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.

Genome-wide identification, characterization, and expression analysis of m6A readers-YTH domain-containing genes in alfalfa.

Eukaryotic messenger RNAs (mRNAs) are often modified with methyl groups at the N6 position of adenosine (m6A), and these changes are interpreted by YTH domain-containing proteins to regulate the metabolism of m6A-modified mRNAs. Although alfalfa (Medicago sativa) is an established model organism for forage development, the understanding of YTH proteins in alfalfa is still limited. In the present investigation, 53 putative YTH genes, each encoding a YT521 domain-containing protein, were identified within the alfalfa genome. These genes were categorized into two subfamilies: YTHDF (49 members) and YTHDC (four members). Each subfamily demonstrates analogous motif distributions and domain architectures. Specifically, proteins encoded by MsYTHDF genes incorporate a single domain structure, while those corresponding to MsYTH5, 8, 12, 16 who are identified as members of the MsYTHDC subfamily, exhibit CCCH-type zinc finger repeats at their N-termini. It is also observed that the predicted aromatic cage pocket that binds the m6A residue of MsYTHDC consists of a sequence of two tryptophan residues and one tyrosine residue (WWY). Conversely, in MsYTHDF, the binding pocket comprises two highly conserved tryptophan residues and either one tryptophan residue (WWW) or tyrosine residue (WWY) in MsYTHDF.Through comparative analysis of qRT-PCR data, we observed distinct expression patterns in specific genes under abiotic stress, indicating their potential regulatory roles. Notably, five genes (MsYTH2, 14, 26, 27, 48) consistently exhibit upregulation, and two genes (MsYTH33, 35) are downregulated in response to both cold and salt stress. This suggests a common mechanism among these YTH proteins in response to various abiotic stressors in alfalfa. Further, integrating qRT-PCR with RNA-seq data revealed that MsYTH2, MsYTH14, and MsYTH16 are highly expressed in leaves at various development stages, underscoring their potential roles in regulating the growth of these plant parts. The obtained findings shed further light on the biological functions of MsYTH genes and may aid in the selection of suitable candidate genes for future genetic enhancement endeavors aimed at improving salt and cold tolerance in alfalfa.

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.

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.

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.

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.

Candidate Genes for Salt Tolerance in Forage Sorghum under Saline Conditions from Germination to Harvest Maturity.

To address the plant adaptability of sorghum (Sorghum bicolor) in salinity, the research focus should shift from only selecting tolerant varieties to understanding the precise whole-plant genetic coping mechanisms with long-term influence on various phenotypes of interest to expanding salinity, improving water use, and ensuring nutrient use efficiency. In this review, we discovered that multiple genes may play pleiotropic regulatory roles in sorghum germination, growth, and development, salt stress response, forage value, and the web of signaling networks. The conserved domain and gene family analysis reveals a remarkable functional overlap among members of the bHLH (basic helix loop helix), WRKY (WRKY DNA-binding domain), and NAC (NAM, ATAF1/2, and CUC2) superfamilies. Shoot water and carbon partitioning, for example, are dominated by genes from the aquaporins and SWEET families, respectively. The gibberellin (GA) family of genes is prevalent during pre-saline exposure seed dormancy breaking and early embryo development at post-saline exposure. To improve the precision of the conventional method of determining silage harvest maturity time, we propose three phenotypes and their underlying genetic mechanisms: (i) the precise timing of transcriptional repression of cytokinin biosynthesis (IPT) and stay green (stg1 and stg2) genes; (ii) the transcriptional upregulation of the SbY1 gene and (iii) the transcriptional upregulation of the HSP90-6 gene responsible for grain filling with nutritive biochemicals. This work presents a potential resource for sorghum salt tolerance and genetic studies for forage and breeding.

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.

Transcriptomic analysis reveals that methyl jasmonate confers salt tolerance in alfalfa by regulating antioxidant activity and ion homeostasis.

Introduction: Alfalfa, a globally cultivated forage crop, faces significant challenges due to its vulnerability to salt stress. Jasmonates (JAs) play a pivotal role in modulating both plant growth and response to stressors. Methods: In this study, alfalfa plants were subjected to 150 mM NaCl with or without methyl jasmonate (MeJA). The physiological parameters were detected and a transcriptomic analysis was performed to elucidate the mechanisms underlying MeJA-mediated salt tolerance in alfalfa. Results: Results showed that exogenous MeJA regulated alfalfa seed germination and primary root growth in a dose-dependent manner, with 5µM MeJA exerting the most efficient in enhancing salt tolerance. MeJA at this concentration elavated the salt tolerance of young alfalfa seedlings by refining plant growth, enhancing antioxidant capacity and ameliorating Na+ overaccumulation. Subsequent transcriptomic analysis identified genes differentially regulated by MeJA+NaCl treatment and NaCl alone. PageMan analysis revealed several significantly enriched categories altered by MeJA+NaCl treatment, compared with NaCl treatment alone, including genes involved in secondary metabolism, glutathione-based redox regulation, cell cycle, transcription factors (TFs), and other signal transductions (such as calcium and ROS). Further weighted gene co-expression network analysis (WGCNA) uncovered that turquoise and yellow gene modules were tightly linked to antioxidant enzymes activity and ion content, respectively. Pyruvate decar-boxylase (PDC) and RNA demethylase (ALKBH10B) were identified as the most central hub genes in these two modules. Also, some TFs-hub genes were identified by WGCNA in these two modules highly positive-related to antioxidant enzymes activity and ion content. Discussion: MeJA triggered a large-scale transcriptomic remodeling, which might be mediated by transcriptional regulation through TFs or post-transcriptional regulation through demethylation. Our findings contributed new perspectives for understanding the underneath mechanisms by which JA-mediated salt tolerance in alfalfa.

Identification and evolution analysis of YUCCA genes of Medicago sativa and Medicago truncatula and their expression profiles under abiotic stress.

The YUCCAs (YUC) are functionally identified flavin-containing monooxidases (FMOs) in plants that act as an important rate-limiting enzyme functioning in the auxin synthesis IPA (indole-3-pyruvic acid) pathway. In this study, 12 MsYUCs and 15 MtYUCs containing characteristic conserved motifs were identified in M. sativa (Medicago sativa L.) and M. truncatula (Medicago truncatula Gaertn.), respectively. Phylogenetic analysis revealed that YUC proteins underwent an evolutionary divergence. Both tandem and segmental duplication events were presented in MsYUC and MtYUC genes. Comparative syntenic maps of M. sativa with M. truncatula, Arabidopsis (Arabidopsis thaliana), or rice (Oryza sativa L.) were constructed to illustrate the evolution relationship of the YUC gene family. A large number of cis-acting elements related to stress response and hormone regulation were revealed in the promoter sequences of MsYUCs. Expression analysis showed that MsYUCs had a tissue-specific, genotype-differential expression and a differential abiotic stress response pattern based on transcriptome data analysis of M. sativa online. In addition, RT-qPCR confirmed that salt stress significantly induced the expression of MsYUC1/MsYUC10 but significantly inhibited MsYUC2/MsYUC3 expression and the expression of MsYUC10/MsYUC11/MsYUC12 was significantly induced by cold treatment. These results could provide valuable information for functional analysis of YUC genes via gene engineering of the auxin synthetic IPA pathway in Medicago.

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.

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

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

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.

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.

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.