Impacts of space mutation and endophyte on the drought and salt stress tolerance of perennial ryegrass

Aims This study revealed whether endophyte or space mutation have effects on the resistance of perennial ryegrass to drought and salt stress and whether space mutation have impacts on endophyte function. Methods Growth performance, phytohormone contents, stoichiometry of C, N and P, and Na + and K + ion transport of endophyte-infected (E+) and endophyte-free (E-) plants, including space mutation and wild type plants, were evaluated under four different treatments (control, drought, salt, and drought combined with salt stress). Results The results showed that stress treatments signicantly inhibited (P<0.05) the growth of perennial ryegrass and signicantly changed (P<0.05) SA contents, signicantly increased (P<0.05) ion transport from underground to aboveground and changed stoichiometry of C, N and P. Endophytes only signicantly improved (P<0.05) host growth in wild type plants, and had different effects on CTK, GA and SA contents. Endophytes had little effects on regulating the rational distribution of ions and stoichiometry of C, N and P in both mutant and wild type plants, and its role was inconsistent. Space mutation signicantly improved (P<0.05) host growth under some stress, signicantly reduced (P<0.05) IAA, ABA, GA and SA contents, Na + / K + had different effects on stoichiometry of C, N and P which depends on treatments. Conclusions Plants regulate stoichiometry of C, N and P and ion transport in response to environmental changes. Either endophyte and space mutation had benecial effects on the stress tolerance of plant only under some treatments. Space mutation did not affect endophyte function.


Introduction
Perennial ryegrass (Lolium perenne) is a globally important forage and turf grass species owing to its desirable agronomic performance in temperate climates. Extensive studies have con rmed that the Epichloë festucae var. lolii endophyte is an essential component of perennial ryegrass cultivation systems owing to its capacity to enhance agronomic performance (Latch et al. 1984;Johnson et al. 2013). E. festucae var. lolii mutualistically interact with perennial ryegrass because it provides major tness enhancements and protection against both biotic and abiotic stresses Young et al. 2013;Ross 2016). Especially about stress tolerance, there were many reports about endophytes have increased host tolerance to drought and salt stress (Wang et al., 2020;Cheplick et al., 2000;Malinowski et al., 2000). Furthermore, these results indicated that the complicated interaction between endophyte and host were also in uenced by genotype, environment and some other factors.
Most of the commercial perennial ryegrass cultivars widely used in China are imported from overseas. Several approaches to breed new perennial ryegrass varieties with domestic adaptive traits and promote seed production have been implemented. Space mutation has been applied to plant breeding in the past 30 years in China and a number of new cultivars or selections of rice, wheat, maize, green pepper, and watermelon were developed using this method (Liu et al. 2007; Paran et al. 2007). To overcome some of the limitations of traditional introgression and selective breeding, some endophyte-infected (E+) and endophyte-free (E-) perennial ryegrass seeds were treated with space mutation.
Mutant germplasm resources were established, some growth parameters of individual plants were measured, and some plants were selected for further evaluation and utilisation. In order to further select individuals with strong stress tolerance, it is necessary to further evaluate their performance and physiology response under different stress conditions. As these mutant germplasms included endophyte associations, the effects of space mutation on function of endophyte in perennial ryegrass and their interactions were also need clarify. Hopefully, this clari cation will help us to understand the effects of these two different breeding methods including endophyte and space mutation and make clear how to utilize them.
Thus, in the present study, the further evaluation of the stress resistance of the E+ and E-plants was conducted. The objectives of the present study were to 1) reveal whether endophyte or space mutation have effects on host stress resistance, 2) identify whether space mutations have impacts on endophyte function, and 3) understand the physiological factors of the underlying stress tolerance of perennial ryegrass for the breeding of stress-tolerant cultivars.

Materials And Methods
Plants materials Seeds . 2020 a,b). The seeds from endophyte-infected (E+) and endophyte-free (E-) sub-populations were marked and stored at 4°C. Two hundred seeds per E+ and E-population were carried using "Shenzhou11" spaceship in October, 2016. These seeds were marked as space mutation (SPE+ and SPE-) and stored at 4°C. In April 16, 2017, these space mutation seeds and wild type seeds without space mutation (UE+ and UE-) were planted in experimental eld blocks at the Yuzhong Experimental Station of Lanzhou University. The growth and morphology of all the plants were evaluated, and the seeds were harvested and stored at 4°C.

Experimental design
In May 2019, the well lled, healthy-looking E+ and E-seeds harvested from SPE+, SPE-, UE+ and UE-were planted in plastic trays (30 cm × 25 cm × 8 cm) lled with 1.5 kg soil (commercial ne sandy soil, Lanzhou) that had been sterilized in an oven at 130 ℃ for 30 min. Five rows with 10 seeds were planted per tray at a depth of 1 cm. Four trays per seed lot were prepared and placed in a temperature-controlled greenhouse (18℃ -24℃) with 10 h of illumination per day on the Yuzhong campus of Lanzhou University. After the plants produced four tillers, the endophyte viability in each seedling was determined using microscopic examination of the host leaf sheath pieces after they had been stained with aniline blue (Nan 1996). The seedlings germinated from E+ seeds with longitudinally-orientated hyphae of the endophyte were marked as E+ and the seedlings germinated from E-seeds without hyphae were marked as E-. The marked E+ or E-seedlings were transplanted into round pots (upper diameter -13 cm × lower diameter -10 cm × height -11 cm) containing the same amount of media (sterilised commercial vermiculite and black soil with a w/w ratio 1:3). Each pot had only one seedling and an equal initial water treatment. After one month of stabilisation with same irrigation, four different treatments were established. The pots were weighed and watered to maintain the appropriate relatively soil moisture content (RSMC), and they were irrigated with the same volume of 250 mmol NaCl before treatment to keep the conditions as following: CK (45% RSMC), drought stress treatment (D, 15% RSMC), salt stress treatment (S, 45% RSMC with 250 mmol NaCl), and drought combined with salt treatment (DS,15% RSMC with 250 mmol NaCl). Each treatment had eight replicates for SPE+, SPE-, UE+, and UE-, and they were randomly placed in a greenhouse that is maintained at a constant condition (temperature: 25 ± 2 ℃, humidity: 42 ± 5%). After another month of growth, the plants were destructively harvested for evaluation.
After 28 days of growth, plant height and tiller number of each plant were recorded. The whole plants were then carefully removed from the pots, washed with distilled water, and dried on lter paper. All harvested plants were separated into roots and shoots, and their fresh weights were recorded. Dry weight was obtained after oven-drying the tissue at 60°C until a constant weight was reached. After weighing, the plant materials were ground twice using a mixer mill (Retch 400MM, German) to analyse for ion contents and nutrient elements. Na + , K + , and Ca 2+ ion contents were analysed using atomic absorption spectrometry (M6AA system, Thermo, USA) after mineralisation in a mixture of acids (Hanway and Heidel 1952). Based on these results, the ratios of Na + / K + and S K, Na were calculated as follows: S K, Na =(aboveground K + / Na + )/(underground K + / Na + ) (Flowers and Yeo 1988).
Carbon (TC) content was determined using the K 2 CrO 7 oxidation method (Tanveer et al. 2014). Total nitrogen (TN) and total phosphorus (TP) contents were determined following digestion with sulfuric acid (H 2 SO 4 ) at a temperature of 420°C, and the concentrations in the digested solutions were determined using ow injection analysis using a FIAstar  Table S1). Based on these effects, signi cant differences between single factors (stress treatment, endophyte and mutation) were assessed using the least signi cant difference (LSD) test at P<0.05, generated from one-way analysis of variance (ANOVA) based on separated dataset. Statistical signi cance was set at 95% con dence level.

Discussion
Previous studies revealed that endophytes improve host tolerance to stress through a variety of morphological and The effects of stress and endophyte on the Na + /K + transport of perennial ryegrass In the present study, compared with the control, Na + ion content signi cantly increased (P< 0.05), whereas K + ion content signi cantly decreased (P< 0.05) under stress treatments. Na + / K + and S K, Na also signi cantly increased (P< 0.05) under stress treatments. Ca 2+ levels did not change in most plants. Accumulation of inorganic ions, such as Na + and K + , is one of the mechanisms for osmotic adjustment in plants during stress response (Shabala et al. 2011). This is very important for alleviating host damage because Na + accumulation in plant cells causes extreme damages by inhibiting enzymes, disrupting K + acquisition, inhibiting K + -dependent metabolic processes, and causing oxidative stress (Pan et al. 2016;Zhu 2001). Maintaining constant intracellular K + and Na + balance is essential for metabolic processes in cells and is crucial for plant adaptation in response to saline environments (Zhu 2003 The effects of stress and mutation on stoichiometry of C, N and P of perennial ryegrass In the present study, C and P contents decreased under stress, whereas N content increased. C, N, and P are essential elements, and the tissue elemental stoichiometry has a mechanistic linkage with the growth rate of the organism. The growth rate hypothesis proposes that higher growth rates are associated with lower C/N, C/P, and N/P ratios (Hessen et al. 2007). N availability can stimulate phosphatase activity in the roots (Fujita et al. 2010), which could potentially promote P uptake. P is required to meet the protein synthesis demands for increased growth rates (Hessen et al. 2007). Epichloë endophytes also adjusted C, N, and P contents and C/N and C/P ratios to increase host growth (Song et  Alkaloid production in these individual plants will be evaluated as soon as possible in the near future to provide a more comprehensive understanding for selection and breeding of these materials. The effects of space mutation on perennial ryegrass Space mutations result in abundant and non-directional mutations, which create genetic variability to improve various complicated traits in plants. Space-induced mutation breading is an effective way to breed new varieties and enhance genetic diversity (Liu et al. 2008). In the present study, space mutation had effects on plant performance as space mutation increased tiller number and underground biomass, plant K + and Ca 2+ contents, underground C content, and underground ratio of C/N; however, it reduced plant phytohormone contents, aboveground Na + content, Na + /K + , and aboveground C/N ratio under some treatments. The mutant individuals provided new methods and resources for perennial ryegrass breeding with strong stress tolerance. Using space-induced radiation, a number of advantageous mutations, which were used to make a breakthrough in most desired crop yield, were also achieved. China has produced 41 varieties developed through space-induced mutation breeding of various crop species such as rice, wheat, cotton, sesame, pepper, tomato, and alfalfa (Liu et al. 2008). However, space-mutation approach results in abundant, nondirectional mutations (He et al. 2006). This breeding methodology needs to be followed by many studies, such as material selection, molecular screening of mutants, and earlier generation identi cation of quality characters to successfully breed new varieties. We should continue to select excellent individual plants from the second generation of these perennial ryegrass germplasms with space mutations and conduct characterisation and genetic analysis in combination with molecular techniques. There are no reports on the effects of space mutations on Epichloë endophytes. In the present study, space mutations did not affect endophyte function. Epichloë endophyte, which lived inside E+ seeds, went through space-induced radiation, possibly having radiation mutations in the genome. Studies on endophyte isolation from E+ mutation plants and its characteristics are in progress to reveal the mutation site and mechanism.

Conclusion
We concluded that plants went through signi cant variations in physiology and regulate stoichiometry of C, N and P and Na + /K + transport in response to environmental changes. Both endophyte and space mutations improved perennial ryegrass growth. However, either endophyte and space mutations had bene cial or harmful effects on regulating the rational distribution of ions and stoichiometry of C, N and P and the adjustment varied with treatments. Space mutations did not affect endophyte function. These results suggest that the selection of mutant germplasm for cultivation may only depend on individual plants with excellent performance.

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