Analysis of a radiation-induced dwarf mutant of a warm-season turf grass reveals potential mechanisms involved in the dwarfing mutant

Zoysia matrella [L.] Merr. is a widely cultivated warm-season turf grass in subtropical and tropical areas. Dwarf varieties of Z. matrella are attractive to growers because they often reduce lawn mowing frequencies. In this study, we describe a dwarf mutant of Z. matrella induced from the 60Co-γ-irradiated calluses. We conducted morphological test and physiological, biochemical and transcriptional analyses to reveal the dwarfing mechanism in the mutant. Phenotypically, the dwarf mutant showed shorter stems, wider leaves, lower canopy height, and a darker green color than the wild type (WT) control under the greenhouse conditions. Physiologically, we found that the phenotypic changes of the dwarf mutant were associated with the physiological responses in catalase, guaiacol peroxidase, superoxide dismutase, soluble protein, lignin, chlorophyll, and electric conductivity. Of the four endogenous hormones measured in leaves, both indole-3-acetic acid and abscisic acid contents were decreased in the mutant, whereas the contents of gibberellin and brassinosteroid showed no difference between the mutant and the WT control. A transcriptomic comparison between the dwarf mutant and the WT leaves revealed 360 differentially-expressed genes (DEGs), including 62 up-regulated and 298 down-regulated unigenes. The major DEGs related to auxin transportation (e.g., PIN-FORMED1) and cell wall development (i.e., CELLULOSE SYNTHASE1) and expansin homologous genes were all down-regulated, indicating their potential contribution to the phenotypic changes observed in the dwarf mutant. Overall, the results provide information to facilitate a better understanding of the dwarfing mechanism in grasses at physiological and transcript levels. In addition, the results suggest that manipulation of auxin biosynthetic pathway genes can be an effective approach for dwarfing breeding of turf grasses.


Results
Morphological characteristics of the dwarf mutant. When growing under the greenhouse conditions for 3 years, the mutant plants had shorter stems, wider leaves, lower canopy height and a deeper green color than the WT plants ( Fig. 1A-C). The average plant height of the mutant was 69% shorter than the WT plants and showed 44% of reduction of blade length and internode length ( Table 1, Fig. 2A-E). These phenotypic data confirm the dwarf characteristics in the mutant and suggest that the mutant carries stable mutation(s) for dwarfing. In addition, the anatomical structures of the mutant and WT were consistent with the results of morphological characteristics. In the cross section of leaves, the mutants had longer blade width and thickness than WT (Table 1, Supplementary Fig. 1 and Supplementary Fig. 2).
Physiological changes in the dwarf mutant. Plant physiological and biochemical variations are often used to characterize plant mutations 47 . In this study, we measured the activities of antioxidant enzymes (CAT, G-POD, and SOD), soluble protein, chlorophyll, electric conductivity, and lignin content in young leaves from both the WT and the mutant. The activities of G-POD and SOD significantly increased by 20% in the mutant (p = 0.05), while CAT showing slightly higher activity ( Fig. 3A-C). The mutant and the WT plants had similar soluble protein contents (Fig. 3D). For the content of chlorophyll (Chl), both Chl a and b contents increased and the Chl b increased significantly in the mutant, and the ratios of Chl a to Chl b in the mutant and the WT were 2.83 and 3.36, respectively (Fig. 3E). The increased chlorophyll content was consistent with the greener leaves observed in the dwarf mutant. For relative conductivity, the conductivity of the mutant sample was twice as much as that of the WT (Fig. 3F). The containment of lignin in the mutant compared to that of in the WT plants reduced from 24 to 19% in leaves and increased from 16 to 21% in stem (Fig. 3G). These physiological changes provide additional evidence to verify that the dwarf mutant is different from the WT plants.
Phytohormone content changes in the dwarf mutant. Several hormones can cause plant dwarfing 31 .
The concentrations of GA and BR are often considered to be the main cause of dwarf plants 48 . To better understand the involvement of phytohormone in dwarf mutant, we measured the contents of four major endogenous hormones in leaves of the mutant and the WT plants. The contents of GA 3 and BR in the mutant did not showed statistic differences from the WT plants, suggesting that they may not play a major role in leading to the dwarf mutant. In contrast, the contents of both ABA and IAA were decreased in the mutant. A significant IAA reduction of 26% was detected in the leaves of the mutant (Fig. 3H). The reduced IAA and ABA contents are likely responsible for the dwarfing in the mutant.
Transcriptomic analysis of the mutant. A total of 50.1 Giga base (Gb) clean data, up to 27 million reads for each sample, were obtained (Supplemental Table 1). The clean reads (QC > 30) were mapped to the reference genome sequences of Z. matrella. The alignment efficiency of the clean reads to the reference genome was between 77.3 and 80.2%, indicating that the data was suitable for further analysis. By aligning to the reported reference genome (https ://zoysi a.kazus a.or.jp/), 1875 transcripts showed no hits and were mostly unannotated (Supplemental Table 2). These no hits may represent the genome specificity of the two genotypes that we used for    (Supplementary Fig. 4A). Of the annotated reads, 26,440 showed 60-80% and 23,334 had 80-100% similarities to the known genes (Supplementary Fig. 4B). To study the sequence conservation of Z. matrella in other plant species, we analyzed the species distribution in the NR database. The closest species was Setaria itallica, with 24,521 genes (40.0%) matched. The second closest reference species was Zea mays, which showed 18.1% homology with Z. matrella ( Supplementary Fig. 4C). The GO classification system was used to assess possible functions of the predicted genes. A total of 46,552 unigenes were assigned to 54 GO terms in three main GO ontologies. The top three frequently identified unigenes classified in the cellular component included 'Cell part' (32,677), 'cell' (32,475), and 'organelle' (28,696). 'Binding' (21,012), 'catalytic activity' (20,715), and 'transporter activity' (2769) were the top three GO terms in the molecular function. The top three GO terms in the biological processes were 'Metabolic process' (26,108), 'cellular process' (23,233), and 'single-organism process' (19,437) (Supplementary Fig. 5).
To reveal the function of the 360 DEGs, we assigned the DEGs to the GO terms. A total of 224 unigenes were assigned for 39 GO terms. The percentage of DEGs genes in some GO terms is significantly different from that of all genes, such as 'reproductive process' , 'signaling' , 'multi-organism process' , 'reproduction' , 'extracellular region' , 'membrane-enclosed lumen' , 'electron carrier activity' , and 'structural molecule activity' , suggesting that these DEGs may have involved in the dwarf mutant (Fig. 5).

RT-qPCR analysis of dwarf-related DEGs.
RT-qPCR assays were performed for 12 dwarf-related unigenes from auxin pathway, BR pathway, gibberellin pathway, and cytoskeleton. The RT-qPCR results were consistent with those from RNA-sequencing analysis, although the exact fold change varied between the two techniques (Fig. 7). The results validated the DEGs identified in the RNA-sequencing analyses.

Discussion
The dwarf mutant is phenotypically stable. A stable dwarf mutant is of importance for dwarf plant breeding in Z. matrella. After three years' propagation and observation, the mutant remained its dwarf trait, suggesting that this dwarf mutant is stable at least during clonal propagation. Since the mutant barely produced any seeds, we could not exam the next generation seedlings to determine whether genetic or epigenetic change(s) was responsible for the mutant.
Bulliform cells form a special structure of the leaf epidermis of gramineous plants, which is one of the reference indicators for drought tolerance 49 . Both the dwarf mutant and WT had similar bulliform cells at the veins (Supplementary Fig. 1), suggesting the mutant is likely not defective in drought resistance. Chl a/b ratio is an important indicator of plant shade tolerance 50 . The decreased Chl a/b ratio in the mutant not only makes the greener leaves of the dwarf mutant more attractive than the WT leaves but also indicates that shade tolerance www.nature.com/scientificreports/ of the mutant may have been changed and likely enhanced. As Z. matrella is asexually propagated into the lawn through stolon, we believe that the 60 Co-γ-induced dwarf mutant is an invaluable material for research and turf industrials due to its slow growth for a potential to reduce the cost for mowing, attractive greener leaves, and a high potential of shade tolerance 51,52 .
Antioxidant enzymes activity and other dwarf-related physiology. Of physiological and biochemical characteristics, the dwarf mutant exhibited differences, particularly in the activity of antioxidant enzymes (POD and SOD) (Fig. 3B,C), from the WT grass. Some of these differences were well correlated with the phenotypic changes in the mutant. For example, the activity of POD is often negatively correlated with stem elongation because of its impact on distribution of IAA in plants 53 . Therefore, there was no surprise that the increased POD was likely responsible, at least in part, for the dwarf mutant by enhancing oxidation of endogenous IAA decomposition. It was also possible that the accelerated formation of the xylem in the dwarf mutant caused an increase of electrolyte content in the leaves of the mutant, which is reflected by variation of relative conductivity.
The DEGs of plant hormones in the dwarf mutant. Plant hormones are multifunctional in plant morphogenesis, growth, and metabolism 54 . Studies have shown that plant dwarfing mutations are closely related to GA, BRs, or IAA 55 . In rice and wheat, GA is one of the key factors that contribute to plant height [56][57][58] . Many dwarf mutants identified were related to BR biosynthetic and signaling pathways [59][60][61] . IAA is also a major determinant of plant growth by activating cell elongation 62 . IAA regulates plant growth mainly through biosynthesis and transportation 63,64 . The biosynthesis of IAA is composed of two pathways: the tryptophan (Trp)-independent and Trp-dependent pathways 65 . Cytochrome P450s catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis. The double mutant cyp79b2cyp79b3 (cytochrome P450s were silenced) showed that the hypocotyls of seedlings were shortened, the plants were dwarfing, and the content of auxin was reduced 66 . PIN-FORMED (PIN) proteins are considered to be auxin efflux carriers, which play an important regulatory role in the polar transport of auxin 67,68 . The abnormal expression of key genes for auxin transport can cause plant dwarfism. Several DEGs related to auxin were down-regulated in the dwarf mutant lines of wheat induced by γ-ray irradiation, such as IAA synthesis and transport related genes 69 . In this study, the decreased IAA content and the IAA-related DEGs revealed that the dwarf mutant of Z. matrella was closely related to the IAA pathway. Biochemically, IAA synthesis in the dwarf ABA plays pleiotropic physiological roles in growth and exhibits potential relationship with plant dwarfing 68 . Cytochrome P450 enzymes are involved in degradation of ABA through 8ʹ-hydroxylase 70 . In this study, the downregulated cytochrome P450 expression in ABA pathway could result in ABA accumulation due to a decreased ABA degradation, leading to the dwarf phenotype in the mutant.
The BR biosynthesis and signal transduction pathway were only recently clarified 71,72 . BR can control plant growth through cell elongation 73 . T-complex protein 1 (TCP1) regulates the synthesis of BR by regulating the expression of the dwarf4 gene. The loss of the TCP1 gene function will lead to dwarf Arabidopsis plants and shorter hypocotyls that can be recovered by exogenous BR 74 . In this study, although the content of endogenous BR in the dwarf mutant did not show a significant change, a DEG annotated to TCP1 in the BR pathway is downregulated. Therefore, it was possible that BR was involved in the formation of the dwarf mutant.

Cell wall and plant dwarfism.
Cell wall is a three-dimensional network composed of polysaccharides, proteins, and other components formed by the intercrossing of cellulose and pectin 75 . The degree of cell wall extension can affect plant height. A mutation in the cellulose synthesis gene can also cause dwarfism 75 . The most widely-studied cellulose biosynthetic enzymes are Cellulose Synthase (CESA). In Arabidopsis, CESA1, CESA3, and CESA6 are essential for primary wall synthesis, preferentially expressed in swollen tissues, and their mutants exhibit an extremely dwarfed phenotype or lethal at seedling stage 76 . Similarly, in this study, the down-regulated CESA1 observed provides additional evidence to verify the dwarf mutant at gene expression levels.
Expansin is a family of nonenzymatic proteins in the plant cell wall that are primarily involved in cell growth, elongation, and several cell wall modifications 77 . Increased expansin relaxes the cell wall tension and enables a higher degree of cell wall extension for taller plants. For example, the overexpression of OsEXP4 (an expansin gene) in transgenic rice resulted in taller plants by 12% 78 . In this study, thirteen down-regulated DEGs of expansin in the dwarf mutant suggested a potential of a reduced expansin accumulation and a reduced plant height.   81 . In this study, higher lignin content in stem was detected in the dwarf mutant of Z. metralla, which is consistent with the findings of similar research in rice 82 . However, it is very interesting that in leaves approximately 20% reduction of acetyl bromide lignin levels was found in dwarf mutant leaves.

Conclusions
A dwarf mutant of Z. matrella induced by gamma radiation was identified from callus regeneration. The significance of the transcriptome data, as well as the morphological and physiological characteristics, suggests that IAA transportation and expansin likely contribute to the major differences in the dwarfism of Z. matrella. This dwarf mutant provides a unique material to study plant growth and dwarf mechanism in turf grasses.

Materials and method
Plant material. In our previous study 1 , different doses of 60 Co γ irradiation (0, 5, 10, 20, 40, 80, 100, 150, 200, 250, and 300 Gy) were used to study the impact of radiation exposure on callus growth and regeneration. The induced plants from the survival calluses after 60 Co γ irradiation were transferred to a greenhouse to select desirable mutants. One dwarf line obtained under 100 Gy irradiation was selected according to leaf size, internode length, and leaf color. The mutant was transplanted, expanded, and pruned to observe its genetic stability. The regenerated plants from the calluses without irradiation were used as a wild type (WT) control.
Morphological evaluation in the greenhouse. After  . G-POD activity was calculated in terms of optical density of guaiacol oxidized min −1 g −1 fresh weight. The SOD activity was determined using a modified version of the method presented in Giannoplitis and Ries 86 , incubating 100 μl of the supernatant in 3 ml reaction solution, containing 50 mM phosphate buffer (pH 7.8, 0.1 mM EDTA), 130 mM methionine, 700 μM Nitrotetrazolium blue chloride (NBT), 13 μM riboflavin for 1 h at room temperature under 20 μmol m −2 s −1 fluorescent light. The absorbance was measured at 560 nm. One unit of SOD activity is defined as the amount of enzyme required to inhibit the photoreduction of NBT by 50%. The content of soluble protein was determined using coomassie brilliant blue G-250 87 . In each sample, 15 μl of supernatant was homogenized in 3 ml coomassie brilliant blue G-250 and absorbance was measured at 595 nm after 2 min. At the same time, the standard curve was made from bovine serum albumin. According to the standard curve and the OD 595 value of the sample, the corresponding protein content (mg/g fresh weight) was calculated. Each determination was repeated three times.

Determination of chlorophyll and relative conductivity.
A modified version of the method describe by Tait and Hik was used to measure chlorophyll concentration 88 . Briefly, a total of 0.1 g of fresh leaves sliced into 5 mm pieces were soaked in 8 ml of DMSO for 72 h in dark at 25 °C. Chlorophyll extract was transferred to a cuvette and absorbances at 649 and 665 nm (Abs649 and Abs665) were measured by a spectrophotometer (UV-2550, SHIMADZU, Tokyo, Japan) and chlorophyll content was calculated using the equations: Chl a = 12.19 A 665 − 3.45 A 649 and Chl b = 21.99 A 649 − 5.32A 665 . To determine relative conductivity, 0.1 g of fresh leaves sliced into pieces (< 5 mm) were soaked in 10 ml of deionized water for 2 h in a 32 ℃ water bath. Initial electrical conductivity of the medium (R1) was measured using an electric conductivity meter (DDB-6220, Shanghai LIDA Instrument Factory, China), and the final electrical conductivity (R2) was measured after the extract was autoclaved at 121 °C for 20 min and cooled to room temperature. Ion leakage was calculated as the ratio of R1/R2 89,90 . Determination of endogenous hormones. In total, 0.3 g of fresh leaves per sample were homogenized in 3 ml of extraction solution (80% methanol containing 1 mM of 2,6-Di-tert-Butyl-4-Methylphenol) using a mortar and pestle at 4 °C. The homogenate was then transferred to a 10 ml test tube, and this procedure was repeated twice. The mixture was inoculated for 4 h at 4 °C and was then centrifuged at 3500g for 8 min. The supernatant was collected and the residue was washed with 1 ml extraction solution. The supernatant was purified using a C-18 solid-phase extraction column, dried with methanol through nitrogen, then diluted with Scientific Reports | (2020) 10:18913 | https://doi.org/10.1038/s41598-020-75421-x www.nature.com/scientificreports/ 2 ml sample diluent (PBS, containing 1% gelatin and 1% Tween-20). The contents of four endogenous hormones (i.e., IAA, GA3, ABA, and BR) were detected using Antibody-Based Enzyme-Linked Immunosorbent Assay, as reported by Deng et al. 91 . Absorbance was read at 492 nm on the microplate reader and the data were analyzed using ANOVA and Duncan with SPSS.
Lignin contents. The acetyl bromide method was used to determinate lignin content 92 . 20 mg of dried leaves and stolon collected separately were reacted with 2 ml of 25% (v/v) acetyl bromide in acetic acid, sealed with Teflon lined caps heated at 70 °C in a water bath, and put in a shaking incubator for 30 min. Afterwards, 200 μl of the digesting mixture was transferred to a 10 ml centrifuge tube containing 0.45 ml of 2 M NaOH and 2.5 ml of acetic acid, 0.05 ml of 7.5 M hydroxylamine, as well as 5 ml acetic acid. The absorbance of the supernatant was measured at 280 nm after centrifuging at 3000g for 7 min. Lignin contents were calculated according to the standard curve. The total content of lignin is finally expressed by dividing the weight of lignin by the weight of the dried sample (%).
Total RNA extraction and library construction for analysis. Total  Identification and functional annotation of DEGs. DESeq 97 and Q-value were used to evaluate differential gene expression between the dwarf mutant and the WT. After that, differences in gene abundance between samples were calculated based on the ratio of FPKM values. Significant DEGs were determined as expressed at a P < 0.01), and fold change (FC) > 2 98 .

Quantitative reverse transcription PCR (RT-qPCR) verification.
For qRT-PCR analyses, 1 μg total RNA per sample extracted from leaf tissues was used to synthesize cDNA with the PrimeScript RT Kit (TaKaRa, Japan). We subjected 12 dwarf-related unigenes to RT-qPCR analysis. The primers listed in supplementary Table 3 were designed using an online tool (https ://www.gensc ript.com/tools /PCR-prime rs-desig ner). ZmActin was used as a housekeeping gene. Amplification was performed as follows: denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 10 s. All reactions were performed in triplicates. Products were verified by melting curve analysis. The 2 −ΔΔCt method was used to calculate the relative expression levels of selected genes 99 .