Current Knowledge of Bermudagrass Responses to Abiotic Stresses

Breeding Science Preview This article is an Advance Online Publication of the authors’ corrected proof. doi:10.1270/jsbbs.18164 Note that minor changes may be made before final version publication.

Review

Current knowledge of bermudagrass responses to abiotic stresses

Shilian Huang†1), Shaofeng Jiang†2), Junsong Liang3), Miao Chen*4) and Yancai Shi5)

1) College of Life Sciences, South China Agricultural University, 483 Wushan Road, Guangzhou, Guangdong, 510642, China 2) Key Laboratory of Tumor Immunology and Microenvironmental Regulation, Guilin Medical University, 109 Huan Cheng North 2nd Road, Guilin, Guangxi, 541004, China 3) College of Biology & Pharmacy, Yulin Normal University, 1303 Jiaoyudong Road, Yulin, Guangxi, 537000, China 4) Faculty of Agricultural Science, Guangdong Ocean University, Haida Road #1, Zhanjiang, Guangdong, 524088, China 5) Guangxi Institute of Botany, Chinese Academy of Sciences, 85 Yanshan Town, Guilin, Guangxi, 541006, China

Bermudagrass (Cynodon spp.) is a common turfgrass found in parks, landscapes, sports fields, and golf courses. It is also grown as a forage crop for animal production in many countries. Consequently, bermudagrass has significant ecological, environmental, and economic importance. Like many other food crops, bermudagrass production also faces challenges from various abiotic and biotic stresses. In this review we will focus on abiotic stresses and their impacts on turfgrass quality and yield. Among the abiotic stresses, drought, salinity and cold stress are known to be the most damaging stresses that can directly affect the production of turfgrass worldwide. In this review, we also discuss the impacts of nutrient supply, cadmium, waterlogging, shade and wear stresses on bermudagrass growth and development. Detailed discussions on abiotic stress effects on bermuda grass morphology, physiology, and gene expressions should benefit our current understanding on molecular mechanisms controlling bermudagrass tolerance against various abiotic stresses. We believe that the rapid development of transcriptomics and proteomics, as well as bermudagrass stable transformation technologies will promote the production of new bermudagrass cultivars with desirable tolerance against abiotic stresses.

Key Words: bermudagrass, abiotic stress, stress tolerance, genetic resources, transcriptomics and proteomics, gene editing.

Introduction In field, plants need to counteract various environmental stresses, including nutritional deficiency, drought, salinity, Bermudagrass (Cynodon spp.) is an important turfgrass cold, and/or heat conditions (Huang et al. 2014). These abi- grown commonly in home lawns, sports fields and parks. otic stresses may further be intensified in future, due mainly Due to its strong vegetative growth and its abilities to toler- to the global warming-induced temperature increases and ate trampling, heat and drought stresses (Harlan et al. 1970), water shortages. As a turfgrass, bermudagrass also needs to bermudagrass is also grown as a forage crop to provide ten- tolerate wear injuries caused by crushing, tearing, and der and nutritional food to animals (Huang et al. 2011). shearing (Trenholm et al. 1999). Consequently, maintaining Bermudagrass is a perennial turfgrass indigenous to the proper growth of turfgrass under unfavorable environmental Mediterranean area and has now been found in more than 100 conditions is a great challenge to farmers and requires new countrires, ranging from the tropical to subtropical regions of knowledge and breeding technologies to create new and the world (Harlan and Wet 1969). Numerous bermudagrass improved bermudagrass germplasms with good stress toler- genotypes and hybrids are now used for commercial culti- ances. Current studies on molecular mechanisms controlling vations (Tari et al. 2013) and over one thousand Cynodon plant adaptations to various growth conditions have provid- accessions are now available at the U. S. National Plant ed invaluable information to plant breeders during their Germplasm System. productions of abiotic stress tolerant germplasms (Bonos and Huang 2006, Huang et al. 2014). In this paper, we will Communicated by Hiroshi Ezura focus on recent progress on the identification and validation Received October 17, 2018. Accepted March 21, 2019. of abiotic stress-responsive genes, and the biochemical and First Published Online in J-STAGE on May 28, 2019. molecular mechanisms controlling bermudagrass tolerance *Corresponding author (e-mail: [email protected]) to various stresses. † These authors contributed equally to this work

1 Breeding Science BS Preview Huang, Jiang, Liang, Chen and Shi Effect of nutrient availability leading to higher accumulations of K+ in both bermudagrass shoots and roots. Their results indicated that melatonin has In the southern part of the USA, bermudagrass is often an important role in regulating bermudagrass responses to grown as a forage grass for cattle production (Day and Parker low K+ stress. 1985). Many reports have shown that the yield and quality of bermudagrass are influenced by nitrogen (N) application, Cadmium stress which is also known to improve the efficiency of inorganic nutrient absorption (Day and Parker 1985, Haby 2007, Cadmium (Cd) is a highly toxic and water soluble heavy Wilkinson and Langdale 1974). For example, bermudagrass metal, and can be easily up-taken by plants in fields production with no nitrogen (N) fertilizer application gave (Arasimowicz-Jelonek et al. 2012a, 2012b). Because Cd is only one-ton forage production while application of 400 stable in water, high concentration of Cd in soil can cause pound N per acre gave 8-ton forage production (Burton and serious public concerns about human food safety (Agami DeVane 1952). In addition, efficient N application was re- and Mohamed 2013, Shi et al. 2014b, Uraguchi and ported to increase protein content in bermudagrass forage Fujiwara 2013). Cadmium contamination in soil was also (Burton et al. 1956, Gilbert and Davis 1971, Westerman reported to inhibit plant shoot and root growth, leading to a et al. 1983). It was reported that sufficient N fertilization reduction of crop production (Bhardwaj et al. 2009, Tan et increased the concentrations of N and potassium (K) in al. 2017). Phytoremediation is a method used to remove the aerial forage and root of bermudagrass cv. Tifton 85. It heavy metals from soil through cultivation of specific plants had, however, no significant effect on phosphorus (P) con- (Cunningham and Berti 1993, Salt et al. 1998). centration. Except for K, N fertilization-induced changes of It was reported that bermudagrass biomass, leaf chloro- nutrient compositions and proportions were reported to be phyll content, photosynthetic pigment production and gas limited (Liu et al. 2017). Abundant N fertilization was exchange parameters could all be affected by Cd stress shown to increase shoot and leaf growth, whereas lower (Mukhtar et al. 2013, Xie et al. 2014). Compared with the amounts of N fertilization resulted in reduced shoot growth Cd susceptible bermudagrass ecotypes, the Cd tolerance and forage quality (Heshmati and Pessarakli 2011, Schmidt bermudagrass ecotypes often showed higher net assimila- and Blaser 1969). The length of bermudagrass root was tion rate, higher water usage efficiency, thickened leaves, found to be shorter when less amount of N was applied reduced leaf surface stoma density, and lower water transpi- (Heshmati and Pessarakli 2011). Different N sources have ration rate (Mukhtar et al. 2013, Xie et al. 2014). Under also been reported to have effects on bermudagrass yield heavy metal stresses, a large number of glandular-like struc- and N uptake. The relative efficiency of different N sources tures were found to form on root surfaces. Formation of was shown as in the order of (NH4)2SO4 (AS) > urea- these glandular-like structures on root surface was consid- ammonium-nitrate (UAN) > urea > anhydrous ammonia ered as an adaptive response to Cd stress (Kuo et al. 2005). (AA) > ammonia after passing through a Cold-Flo adapter A total of 39 metabolites in bermudagrass were found to be (ACF) (Westerman et al. 1983). Silveira et al. (2007) re- responsive to Cd stress. These metabolites included some ported that ammonium nitrate (AN) and AS increased amino acids, organic acids, sugars, and fatty acids (Xie et bermudagrass forage yield and N uptake when compared al. 2014). Shi et al. (2014b) reported that application of ex- with urea and UAN in Gallime soil. However, N sources ogenous nitric oxide (NO) donor or hydrogen sulfide (H2S) made no clear effect on dry matter (DM) production when donor could induce bermudagrass tolerance to Cd stress. the plants were grown in lilbert soil. The authors speculated that the oxide-activated hydrogen Increasing amount of N, P and K application increased sulfide is essential for inducing Cd tolerance in bermuda total nutrient uptake, total protein content, and P and K con- grass. In a different study, Li et al. (2006) found that over- tents in Coastal bermudagrass forage (Adams et al. 1966). expression of Phytochelatin synthase (CdPCS1) gene in Higher rate of K application was considered to improve turf tobacco also increased the accumulation level of Cd. grass appearance and growth. However, when the amount of K fertilizer exceeded the N/K ratio of 0.5 to 1, no significant Drought stress K effect on bermudagrass growth or root weight was observed (Snyder and Cisar 2000). It was reported that the ratio Bermudagrass is considered to be a drought tolerant species of (4-5):(4-5):1 (N:K:P) could benefit bermudagrass regrowth with genotypic variations (Husmoen et al. 2012). Several while the ratio of 4:1:6 (N:K:P) could enhance bermuda bermudagrass genotypes were found to have deep root sys- grass tolerance to cold stress (Snyder and Cisar 2000). Sul- tems, thick leaf cuticles and smaller stomatal openings fur (S) fertilizer application was reported to increase the (Carrow 1995, 1996, Qian et al. 1997, Zhou et al. 2009). N/S ratio, but had no impact on bermudagrass forage Deep and large root system is a well-known character for yield, N uptake or efficiency of N utilization (Westerman plant drought tolerance (Beard 1989). The capacity of water et al. 1983). More recently, Chen et al. (2017) showed that absorption by root depends largely on root size and spatial application of exogenous melatonin to bermudagrass up- distribution (Carrow 1996, Huang 2000, Huang et al. 2014). regulated the expressions of K+ transport-related genes, Besides, Zhou et al. (2014b) found that the Mediterranean

2 Breeding Science Responses of bermudagrass to abiotic stresses Preview BS bermudagrass evolved a large rhizome system adapting to damage. Several studies have shown that, under drought drought stress. Unlike cold-season grasses, warm-season stresses, accumulations of 16-, 23-, 31- and 40-kDa dehy- grasses can continue to grow their roots in the summer drins in drought tolerant bermudagrass cultivars were differ- (Beard 1973). In regular soils or in acidic soils, the depth of ent from that in the drought sensitive bermudagrass culti- grass roots ranged as bermudagrass cv. Tifway > common vars (Hu et al. 2010, Su et al. 2013). Hybrid bermudagrass bermudagrass or tall fescue cv. Rebel > tall fescue cv. cv. Tifway and regular bermudagrass cv. C299 both showed Kentucky-31 > St. Augustinegrass [Stenotaphrum secundatum a decline in photosynthetic rate, caused by stomatal closure (Walt.) Kuntzel] cv. Raleigh > common centipedegrass during an early drought stress stage (Hu et al. 2009, Huang [Eremochloa ophiuroides (Munro) Hack.] > Meyer’ zoysia- et al. 2014). However, compared with cultivar C299, culti- grass (Zoysia japonica Steud.) (Carrow 1996). Baldwin et var Tifway was able to maintain a higher photosynthetic al. (2006) reported that the root weight of six well-irrigated rate even though the stomatal conductance was decreased to bermudagrass cultivars was significantly greater than that of near zero (Hu et al. 2009). Higher Rubisco enzyme activi- the same cultivars with less irrigation. Leaf wilting is a ma- ties and more stable carbon assimilation proteins in cultivar jor visual phenotype caused by drought stress and several Tifway were found to be responsible for greater carboxyla- other biotic and/or abiotic stresses. Compared with other tion capability (Hu et al. 2009, Huang et al. 2014). Huang et turfgrasses (e.g., centipedegrass, tall fescue and Meyer’ al. (2014) reported that accumulation of reactive oxygen zoysiagrass), bermudagrass has a much lower leaf wilting species (ROS) in plant was significantly induced by drought rate, indicating its better drought tolerance than other grasses. stress and, at the same time, plant has its own strategies Besides, bermudagrass cv. Tifway was reported to have a scavenge ROS. One of the most effective ROS scavenging lower leaf wilting rate than other commonly grown bermuda strategies is to regulate the expression and/or activities of grass (Carrow 1996). To reduce leaf desiccation, plants have antioxidant enzymes, including superoxide dismutase evolved a defense mechanism to control stomatal closure. (SOD), catalase (CAT) and ascorbate peroxidase (APX). Lu By controlling stomatal closure, plants are able to reduce et al. (2008, 2009a) demonstrated that the increased activi- their evapotranspiration rate (ET) (Huang et al. 2014). ties of SOD, CAT, and APX in leaves of mutant bermuda Compared with other turfgrasses, Carrow (1995) indicated grass plants could clearly improve their drought tolerance. that bermudagrass had a lower ET rate than other turf Compared with bermudagrass cv. TifEagle, three somaclon- grasses. In the same report, he also indicated that the ET al variants of triploid bermudagrass showed higher CAT rate of well-irrigated bermudagrass is lower than that of less activity during drought stress, and two variants showed well-irrigated bermudagrass. Consequently, drought toler- higher APX activity during short-term drought stress and ance has a direct effect on bermudagrass forage quality higher SOD activity after long-term drought stress. The (Baldwin et al. 2006). same research group reported in the same year that the ac- Drought stress causes significant physiological changes, tivities of ABA-induced SOD and CAT could be inhibited including photosynthesis and antioxidant metabolism, in by application of ROS scavenger H2O2 or NO (Lu et al. plant. It is generally accepted that greater volume of root 2009b). systems and slower shoot growth allow bermudagrass to Analysis of cDNA libraries constructed using bermuda maintain enough water content and normal cellular function grass cv. Tifway and C299 through suppression subtractive during drought seasons. Several metabolites have been re- hybridization, Zhou et al. (2014a) identified a total of 277 ported to be important for maintaining a proper cell osmotic drought responsive genes. The up-regulated genes identified pressure and reducing water evaporation (Hu et al. 2015a). in that study included those related to drought avoidance For example, under drought stresses, Arizona Common and traits (i.e., CER1 and sterol desaturase), drought tolerance Coastal bermudagrass were shown to have higher levels of traits (i.e., dehydrins, HVA-22-like protein, superoxide dis- free proline, asparagine and valine but lower level of free mutase, dehydro-ascorbate reductase [DHAR], and 2-Cys alanine (Barnett and Naylor 1966). Compared with the peroxiredoxins), and stress signaling (i.e., EREBP-4 like drought susceptible variety Yukon, cultivar Tifgreen had protein and WRKY transcription factors). In a different much higher levels of proline and soluble sugars (Shi et al. study, 120 genes, including a putative delta-pyrroline-5- 2012). Du et al. (2012) studied a hybrid bermudagrass using carboxylate synthetase (ES292694) and a putative MYB17 a gas chromatograph coupled with a mass spectrometer and protein (ES295217), were found to be up-regulated (Kim et determined that the levels of three organic acids, ten amino al. 2009). The putative delta-pyrroline-5-carboxylate syn- acids and seven sugars were significantly up-regulated. In thetase is an essential enzyme for proline synthesis and many recent studies, relative water content and electrolyte MYB17 is known as a stress responsive transcription factor leakage have been used as indications of plant drought tol- (Kim et al. 2009). Analysis of the upstream part of CdDHN4 erance and the degree of cell membrane damage. Accumula- gene in tobacco leaves showed that the promoter of tion of unsaturated fatty acids in membrane lipids was con- CdDHN4 could be regulated by ABA application, or by sidered to reduce cell membrane damage during drought drought or cold treatment. Further study on this gene stresses (Zhong et al. 2011). Besides osmotic regulation, showed that CdDHN4 played an important role in the ABA- dehydrin proteins were also reported to protect membrane dependent signaling pathway induced by drought stress

3 Breeding Science BS Preview Huang, Jiang, Liang, Chen and Shi (Lv et al. 2017). Chen et al. (2015b) showed that over- root mass, root number and length, shoot length, and overall expression of Cdt-NF-YC1 gene in rice not only increased turfgrass quality (Beard 1973, Sladek et al. 2009, Trappe et plant tolerance against drought or salt stress but also in- al. 2011). It was reported that shade had greater effect on creased the sensitivity to ABA treatment. Protein synthesis bermudagrass growth than other turfgrasses (Baldwin et al. and degradation are also important during drought stresses. 2008, Gaussoin et al. 1988). Jiang et al. (2003, 2005) com- Zhao et al. (2011) observed that multiple bermudagrass pared the responses of seashore paspalum and bermudagrass genes were involved in the maintenance of protein metabo- to low light conditions. Their results indicated that under the lism during drought stresses. Among these genes, genes en- low light condition, both plants, especially bermudagrass, coding Chl a-b binding protein, ATP synthase or phospho showed a clear reduction of turf quality, canopy photosyn- ribulokinase were important during photosynthesis, genes thetic rate, normalized difference vegetation index (NDVI), encoding SOD, APX or DHAR were involved in antioxidant chlorophyll a (Chl a) and b (Chl b) contents, total soluble pro defense, and a gene encoding ADP-glucose pyrophosphory tein content, water-soluble carbohydrate content, and CAT lase took part in starch synthesis (Huang et al. 2014). It was and APX activities, suggesting that bermudagrass might be suggested that drought stress responsive genes could be clas- more sensitive to low light stress than seashore paspalum. sified into five different categories: metabolism category (e.g., Trappe et al. (2011) also showed that bermudagrass had a glycine dehydrogenase, aminomethyltransferase, phospho lower tolerance to shade stress than zoysiagrass cultivars. In ribulokinase, short-chain dehydrogenase/reductase, methio- a separate study using 32 different bermudagrass cultivars nine synthase, ADP-glucose pyrophosphorylase, aspartate grown under the reduced light conditions, Gaussoin et al. aminotransferase and beta-1,3-glucanase); energy category (1988) determined that bermudagrass cvs. Boise, No Mow, (e.g., putative aconitate hydratase, succinate dehydrogenase and NM 2-13 were more tolerant to shade than cvs. Arizona (ubiquinone) flavoprotein subunit and glyceraldehyde-3- Common and Santa Ana. Analysis of forage quality, shoot phosphate dehydrogenase); defense category (e.g., dihydro chlorophyll content, root length, and root biomass, Baldwin lipoyl dehydrogenase, SOD, APX and monodehydro et al. (2008) identified 42 bermudagrass cultivars with ascorbate); protein synthesis category (e.g., 30S ribosomal shade tolerance. The authors also indicated that cvs. Cele- protein S1, elongation factor Tu and ribosomal protein bration, TiftNo. 4, TiftNo. 1, and Transcontinental had the L12); and cell growth/division category (e.g., actin- best shade tolerance. In addition, bermudagrass cvs. Aussie depolymerizing factor 2) (Zhao et al. 2011). This classifica- Green, MS-Choice, Princess 77, SWI-1045, SWI-1041, and tion was supported by more recent proteomics studies via SWI-1012 had intermediate tolerance to shade and cvs. MALDI-TOF-MS (Shi et al. 2014a, Ye et al. 2015, 2016). SWI-1014, Arizona Common, Sundevil, SR 9554, GN-1, and Patriot were the most sensitive cultivars to shade stress. Waterlogging stress Burton et al. (1959) reported that, under low-nitrogen conditions, shade stress could cause worse bermudagrass Flooding also has great effect on plant vegetative and repro- growth and biomass production. Schmidt and Blaser report- ductive growth, and dry mass production (Promkhambut et ed that low light intensity could influence bermudagrass al. 2011, Tari et al. 2013). Flooding stress often related to a forage and root growth, and cause an increase of total N, low bermudagrass root growth rate, less accumulation of possibly caused by limited nitrate reduction and inhibition soluble sugar and starch, and induction of SOD and APX of N utilization (Schmidt and Blaser 1969). activities (Li et al. 2015). To investigate the changes of bermudagrass anti-oxidant enzyme activities and carbohy- Wear stress drate content during flooding, Tanet al. (2010) collected and analyzed bermudagrass samples after various submergence Bermudagrass is a common turfgrass in golf course and treatments. Their results showed that increased duration sports fields in the southern region of the USA, and in coun- and/or depth of submergence further enhanced the activities tries with warm climate (Trenholm et al. 1999). High fre- of CAT, SOD, APX, peroxidase (POD), and glutathione re- quencies of human and equipment trafficking often cause ductase (GR) in bermudagrass roots while the accumulation significant wear stress on turf quality (Griffinet al. 2006). It of total soluble carbohydrate and starch in shoots and roots was reported that wear stress could cause severe damages to were decreased. A recent proteomics and metabolomics bermudagrass canopy appearance followed by leaf senes- study showed that the metabolism and dormancy of bermuda cence (Trenholm et al. 1999). Lulli et al. (2012) reported a grass under flooding was significantly decreased (Ye et al. positive correlation between lignin content and plant wear 2015). The authors speculated that these retarded activities tolerance. They have also indicated that starch content in might help bermudagrass to survive during submergence rhizome and stolon, and glucose content in rhizome corre- stress (Ye et al. 2015). lated negatively to turfgrass wear tolerance (Lulli et al. 2012). It is noteworthy that wear tolerance varies signifi- Shade stress cantly among different bermudagrass cultivars. For example, bermudagrass cv. Riviera showed the best recovery of Shade can also affect turfgrass growth, resulting in reduced green canopy after one, two, or three time simulated wear

4 Breeding Science Responses of bermudagrass to abiotic stresses Preview BS treatments per week (Bayrer 2006) or by using the Cady duced by salt stress were influenced by plant tolerance to traffic simulator (Henderson et al. 2005). Trappe et al. salt stress and its ability to retain water. (2008) also showed that bermudagrass cv. Riviera had a supe Salt stress often causes significant reduction of leaf rela- rior tolerance to wear stress compared with other bermuda tive water content (RWC), transpiration rate, leaf net photo- grass cultivars, except hybrid bermudagrass Tifway. In a synthetic rate (Pn), total chlorophyll content, starch level, separate study, Williams et al. (2010) also found that stomatal conductance, and cellular membrane stability bermudagrass cv. Riviera had a similar wear tolerance as (Bizhani and Salehi 2014, Yu et al. 2015). It also reduces K+ cv. Princess 77, but better than all other four cultivars tested concentration but increases Na+ and Cl– concentrations in in that study. Trappe et al. (2011) reported that bermudagrass turfgrass stolon and shoots (Chen et al. 2014a, Dudeck et al. cvs. Rivera and Tifway had better wear tolerance than cvs. 1983, Nadeem et al. 2012). Hu et al. (2012) treated two dif- Princess 77, Tifsport, and three zoysiagrass cvs. El Toro, ferent bermudagrass genotypes with 200 and 400 mM salt Palisades, and Zorro. It was reported that over-seeding and found that the concentrations of malondialdehyde and could reduce bulk density but increase canopy coverage, hydrogen peroxide in mature leaves of the salt-sensitive thatch accumulation, and saturated hydraulic conductivity genotype C198 were higher than that in the salt-tolerant that can protect bermudagrass from wear stresses (Thoms et genotype C43, while the activities of SOD, CAT, APX, al. 2011). Applications of exogenous plant growth regula- DHAR were much higher in the salt-tolerant genotype com- tors (i.e., ethephon, trinexapacethyl (TE), paclobutrazol, pared with the salt-sensitive genotype. Elevated concentra- flurprimidol, flurprimidol plus TE, and ethephon plus TE) tion of CO2 as well as accumulation of soluble sugars, pro- were also found to improve bermudagrass wear tolerance line, and glycine betaine (GB) were found to reduce the (Brosnan et al. 2010). Besides, the depth of crumb rubber negative effects caused by salinity stress (Yu et al. 2015). other than the size of crumb rubber particle were shown to Hu et al. (2015a) also indicated that the reduced shoot improve the wear tolerance (Dickson et al. 2017). length and increased root length might be caused by an increased accumulation of soluble sugars and metabolites, Salt stress important to nitrogen metabolism, and this physiological change could improve root absorption capability and en- Plant can tolerate salt stresses by secreting Na+ out of the hance bermudagrass salt tolerance. Liu et al. (2016a) found cells, slower up-taking of Na+, and translocating Na+ into that sodium nitroprusside (SNP, a donor of NO), could alle- vacuole inside cells through specific mechanisms (Tariet al. viate salt toxicity to bermudagrass by maintaining the sta- 2013). And osmotic-changing and ionspecific are two dis- bility of cell membrane, ion homeostasis, photosynthesis. tinct phases for salt response (Munns and Tester 2008). With increasing levels of salt stress, the expressions of Though bermudagrass is considered as an efficient grass to antioxidant-related genes were up-regulated in young leaves revegetate sodic land, there is a series of morphology, phys- but down-regulated in mature leaves (Hu et al. 2012). iology, molecular changes under salt stress (Singh et al. Through comparative proteomics, 77 differentially expressed 2013). Pessarakli and Touchane (2006) showed that bermuda proteins were identified upon drought and salt stresses. grass shoot and root length were increased in a solution Most of these identified proteins are known to be involved containing 5,000 or 10,000 mg/L NaCl, and decreased when in glycolysis, oxidative pentose phosphate, photosynthesis the NaCl concentration was further increased. Salt stress and redox metabolic pathways. Thirteen of the identified was shown to reduce turf quality, stolon number per plug, proteins were found to be regulated specifically by salt and shoot weight and height, but to increase root number stress (Ye et al. 2016). With the same approach, Shi et al. and length, fresh root weight, and root/shoot length ratio of (2013) identified 12 up-regulated, 20 down-regulated, and salt-tolerant bermudagrass genotypes (Dudeck et al. 1983, four specifically expressed proteins after the polyamines Hu et al. 2012). Shahba (2010a) reported that a positive cor- treatment. Most of these proteins are known to participate in relation between root mass and salinity tolerance in bermuda electron transport and energy pathways. Recently, Hu et al. grass cvs. Tifway, Tifdwarf and Tifgreen. Hu et al. (2012) did (2018) analyzed four small RNA libraries from mock-, a similar work using bermudagrass cvs. Tifway, Tifdwarf, cold-, salt- or cold plus salt-treated bermudagrass plants, Dacca and ‘Khabbal, and found that at five salinity stress and identified 449 miRNAs. Compared to the mock-treated levels, the root number and dry weight of these four culti- plants, 49 and 39 miRNAs were found to be up- and vars were all significantly decreased. Bizhani and Salehi down-regulated after the salt treatment, respectively. (2014) confirmed later that salt stress could indeed decrease Salt stress causes significant changes in plant morpholo- root fresh and dry weights. It is possible that the different gy, physiology and molecular functions. However, these results described above were caused by different salt stress changes can vary greatly among different bermudagrass tolerance in different bermudagrass cultivars and/or the cultivars or phenotypes. Dudeck et al. (1983) compared the salinity treatments used in above studies. Hameed et al. salt tolerance in eight bermudagrass cultivars, and deter- (2010, 2013) examined the root, stem and leaf morphologi- mined that cvs. Tifdwarf and Tifgreen showed the best toler- cal changes using a natural salt stress adaptive population. ance to salt stress, while cvs. Common and Ormond were Their results showed that the morphological changes in- the most sensitive cultivars. Peacock et al. (2003) reported

5 Breeding Science BS Preview Huang, Jiang, Liang, Chen and Shi that bermudagrass cv. Quickstand produced more total protein of 27 kDa (COR27) was identified as a chitinase shoot biomass then cvs. Tifton-10, Tifway, Navy Blue, (Gatschet et al. 1994, 1996). Zhang et al. (2008) reported that GN-1 and TifSport under a salt stress condition. Chen et al. the expression of a 25 kDa dehydrin protein was increased (2014b) evaluated salt stress tolerance in various bermuda in four tested bermudagrass cultivars upon cold stress. In a grass germplasms collected in China. Based on the per more recent study, Hu et al. (2018) identified 62 up-regulated centage of leaf firing and relative shoot weight, the authors and 45 down-regulated miRNAs after comparing a cold- classified the germplasms into four groups. Dudeck et al. treated bermudagrass with a mock-treated bermudagrass. (1983) analyzed four bermudagrass cultivars and found that Variations of cold resistance were also found among dif- cv. Tifway had the best salinity tolerance and cv. Khabbal ferent bermudagrass cultivars. For example, Anderson et al. was the most susceptible cultivar against salt stress. Lu et (1988) indicated that bermudagrass cv. Mldiron was able to al. (2007) developed an in vitro selection method for tolerate –11°C condition while cv. Tifgreen was able to tol- screening salinity tolerant calli on solid medium containing erate –7°C condition in winter. In 2011, Anderson and Wu high concentrations of salt. Through this screening method, reported that bermudagrass cvs. Hardie, Goodwell, Midland the authors were able to regenerate several plants with 99 and Ozark showed better freezing tolerance than the ref- higher salt tolerance than the parental cv. TifEagle. Besides, erence cv. Midland. In that study, cvs. Coastcross, Tifton 85 exogenous polyamine and mowing were reported to increase and Tifton 68 were ranked the most susceptible bermuda salt tolerance of bermudagrass (Shahba 2010b, Shi et al. grass cultivars. The lethal temperature for causing 50% of 2013). the plants to die (LT50) for bermudagrass cv. Midiron and Tifgreen without a pre-cold-acclimation were at –6.5 and Cold stress –3.6°C, respectively. After pre-cold-acclimation, the LT50 of the two cultivars were increased to –11.3 and –8.5°C, re- Low temperature is one of the main factors that limit the spectively (Gatschet et al. 1994). This finding indicates that geographical distribution of bermudagrass. Cold stress was cold-acclimation can be an effective method for cold toler- shown to decrease turf quality, growth, dry weight, tiller ance improvement. It was also reported that the cold- density, leaf size, chlorophyll content, transpiration rate and acclimation treatment could increase the production of RWC. Cold stress could also increase electrolyte leakage, proline, a 25 kDa dehydrin, and abscisic acid (ABA), but de- and malonaldephyde and hydrogen peroxide contents in crease the level of t-zeatin riboside (a kind of cytokine) that plant (Esmaili and Salehi 2012, Fan et al. 2014, Liu et al. was reported to increase the cold tolerance of bermudagrass 2016b). In contrast, cold stress could cause an increase of (Zhang et al. 2011a, 2011b). ABA was found to enhance the root depth and fresh weight (Esmaili and Salehi 2012). In- cold tolerance of bermudagrass by maintaining the cell creases of soluble sugar and proline content, total non- membrane integrity, improving the function of photosystem structural carbohydrates (TNC) content, fatty acid content, II as well as increasing the carbon isotopic fractionation antioxidant enzyme activity (especially CAT and SOD), and (Huang et al. 2017). Cold-acclimation was also reported to metabolite productions (e.g., carbohydrates, organic acids increase sugar and proline accumulations, and enhancing and amino acids) were also considered to be bermudagrass SOD activity to improve the survival rate under cold stress defense strategies against cold stress (Hu et al. 2016a, 2017, conditions (Zhang et al. 2011a). Cold-acclimation was Liu et al. 2016b, Manuchehri et al. 2014, Zhang et al. 2006, shown to induce gene expressions (De los Reyes 2001, Zhu 2011b). Fan et al. (2015) reported that NO played an impor- et al. 2015), leading to better tolerance of bermudagrass to tant role in maintaining cell membrane integrity, regulating cold stress. In addition, exogenous melatonin application was antioxidant enzyme activities, and controlling cold respon- found to improve bermudagrass cold tolerance by increasing sive gene expressions. Several genes, including MnSOD, antioxidant enzyme activities, altering cold responsive Cu/ZnSOD, POD, APX, embryogenesis abundant proteins metabolite productions (e.g., carbohydrates, organic acids (LEA), C-repeat-binding factor/DRE-binding factor (CBF1) and amino acids), and affecting the function of photosystem and chitinase (CynCHT1), have been shown to be respon- II (Hu et al. 2016b). Comparative proteomics allowed the sive to cold stress (De los Reyes et al. 2001, Fan et al. 2014, identification of 51 differentially expressed proteins after a Hu et al. 2017). Through next generation sequencing, Zhu cold and calcium chloride (CaCl2) treatment. Most of these et al. (2015) compared cold-acclimated and non-cold- identified proteins are known to be involved in photosynthe- acclimated bermudagrass, and identified 5867 differentially sis, redox, glycolysis, tricarboxylicacid cycle, oxidative expressed genes. Among these genes, 2181 were down- pentose phosphate pathway, and amino acid metabolisms. regulated while 710 were up-regulated. Several transcript Regulations of amino acid, organic acid, sugar, and sugar factor families (e.g., AP2, bHLH, NAC, auxin-responsive, alcohol productions indicated that application of exogenous WRKY, MYB, bZIP) were also found to be cold stress re- CaCl2 could improve bermudagrass chilling or freezing tol- sponsive. Several cold responsive (COR) proteins have now erance (Shi et al. 2014c). Munshaw et al. (2004) reported been identified in bermudagrass cvs. Midiron and Tifgreen, that applications of moderate amount of salt or use effluent and many of these COR proteins were found to have low water could increase bermudagrass cold hardiness (Zhang molecular weights, ranging from 20 to 28 kDa. One COR et al. 2008). Hu et al. (2016b) reported that application of

6 Breeding Science Responses of bermudagrass to abiotic stresses Preview BS exogenous ethylene precursor 1-aminocyclopropane-1- physiological and biochemical aspects. Numerous studies carboxylic acid (ACC) resulted in a reversed effect on have shown that under various stress conditions, the cell bermudagrass cold tolerance through impacting its anti membrane permeability, photosynthesis rate, antioxidant en- oxidant system and photosystem II function, and altering zyme activity, soluble sugar and protein content of bermuda the expressions of several important genes (i.e., LEA, SOD, grass have changed accordingly (Table 1). But these changes POD-1 and CBF1, EIN3-1, and EIN3-2). Cheng et al. are just a physiological adjustment of the bermudagrass in (2016) also reported that application of exogenous ABA order to adapt to changes in the environment. The upstream mimic 1 (AM1) improved bermudagrass seedling cold tol- mechanism for these changes is not much studied. As a erance via reducing the contents of H2O2, O2-, -OH and non-model plant, when bermudagrass faces different abiotic malondialdehyde, and increasing the activities of glutathi stresses, it needs to be verified that whether its signal trans- one peroxidase, glutathione reductase and superoxide dis- duction pathway is similar to that of model organisms. At mutase. It was reported that late season N fertilization or ap- the same time, the changes in the molecular level of bermuda plication of trinexapac-ethyl (TE), ethephon and freezePruf grass before and after the adversity are still unclear, and the had no clear effect on bermudagrass cold tolerance research results obtained are limited. (Anderson 2012, Munshaw et al. 2010, Richardson 2002). Based on the morphology and physiological data, significant genetic variations have been found in bermudagrass Conclusions and future perspectives genotypes with various tolerance to drought, salinity, and cold stresses. These genotypes have offered researchers Significant progress has been made on our understandings with valuable resources for future studies on bermudagrass of morphology, physiology and molecular mechanisms as- gene function, abiotic and biotic stress responses, and im- sociated with bermudagrass tolerance to salinity, drought provement of bermudagrass production and quality. For ex- and cold stresses, however, the research mainly focuses on ample, Zhou et al. (2013) tested the drought tolerance of

Table 1. Mainly effects and adaptive characteristics of bermudagrass under various stresses Type of Genes confirmed to be Phenotypic effects Adaptive characteristics stresses involved cadmium reduce biomass, leaf chlorophyll content, photosyn- thickened leaves, reduced leaf surface stoma density, CdPCS1 (Li et al. 2006) stress thetic pigment production and gas exchange parame- glandular-like structures form on root surfaces (Kuo ters (Mukhtar et al. 2013, Xie et al. 2014) et al. 2005, Mukhtar et al. 2013, Xie et al. 2014) drought accumulation of osmotic (such as proline, asparagine, deep root systems, a large rhizome system, thick leaf CdDHN4; Cdt-NF-YC1 stress valine and soluble sugars), dehydrin proteins (such as cuticles and smaller stomatal openings (Carrow 1996, (Chen et al. 2015b, Lv et 16-, 23-, 31- and 40-kDa dehydrins) and ROS, decline Huang and Gao 2000, Huang et al. 2014, Zhou et al. al. 2017) in photosynthetic rate (Barnett and Naylor 1966, Hu 2014b) et al. 2009, 2010, Huang et al. 2014, Shi et al. 2012, Su et al. 2013) waterlog- low bermudagrass root growth rate, less accumulation ging stress of soluble sugar and starch, and induction of SOD and APX activities (Li et al. 2015) shade stress a clear reduction of turf quality, canopy photosynthetic rate, chlorophyll a and b contents, total soluble protein content, water-soluble carbohydrate content, and CAT and APX activities (Beard 1973, Sladek et al. 2009, Trappe et al. 2011) wear stress cause severe damages to bermudagrass canopy high lignin content (Lulli et al. 2012) appearance followed by leaf senescence (Trenholm et al. 1999) salt stress reduction of leaf relative water content, transpiration increased exodermis, sclerenchyma, endodermis, rate, leaf net photosynthetic rate total chlorophyll cortex and pith parenchyma in roots; increased stem content, starch level, stomatal conductance, and area, increased epidermis, sclerenchyma thicknesses, cellular membrane stability (Bizhani and Salehi 2014, cortex thickness, increased number and area of Yu et al. 2015) vascular tissue in stem; increased development of vesicular hairs and less affected parenchymatous tissue in leaf (Hameed et al. 2010, 2013) cold stress decrease turf quality, growth, dry weight, tiller density, leaf size, chlorophyll content, transpiration rate and RWC; increase electrolyte leakage, and malonaldephyde and hydrogen peroxide contents in plant (Esmaili and Salehi 2012, Fan et al. 2014, Liu et al. 2016b)

7 Breeding Science BS Preview Huang, Jiang, Liang, Chen and Shi 460 genotypes collected from different climatic zones of synthase2, galactinol synthase1, stachyose synthase, delta Australia, the result showing that genotypes experienced 1-pyrroline-5-carboxylate synthetase 1) and accumulation Mediterranean climates showed better drought tolerance. of osmotic regulator (such as amino acid, sugars, etc.) will However, it may need more time for screening resistant vari maintain cell turgor (Huang et al. 2014, Zhao et al. 2011, eties. From the information present in the article, we know Zhou et al. 2014a). There are also some photosynthesis, mowing and applications of exogenous plant growth regula- carbon fixation and energy supply proteins (such as tors can improve the wear tolerance, and application of ex- RuBisCO-related proteins, ferredoxin-thioredoxin reductase, ogenous ABA mimic 1 (AM1) improved bermudagrass ferredoxin-NADP reductase) activated by osmotic stress seedling cold tolerance but it will increase labor costs. As a (Zhou et al. 2014a). For cold stress, there exists a similar mature technology, transgene would be a better choice for resistance mechanism in the activation of the proteins in- the future bermudagrass breeding. volved in maintenance of cell homeostasis, osmotic adjust- Recent developments of technologies, including tran- ment, ROS scavenging, photosynthesis and energy supply scriptomics, proteomics, and metabolomics let us have a (Chen et al. 2015a, Liu et al. 2016b, Zhu et al. 2015). The deeper understanding of its regulatory mechanisms. In order first step of the resistance to adversity is the perception of to adapt to osmotic stress caused by salt and drought, adversity. As a second messenger, Ca2+ plays an important bermudagrass will form a layer of wax on the leaf surface. role in signal transmission. Different stress can cause differ- The up-regulation of CER1 and sterol desaturase may con- ent calcium signals, generating the so-called Ca2+ signature, tribute to the formation of waxy (Zhou et al. 2014a). Osmotic which can be sensed by Ca2+ sensors (CDPK, CaM (CML) stress has great damage to the cell membrane, dehydrins and and CBL) and then transduced through interacting with late-embryogenesis abundant (LEA) proteins may play the downstream proteins, including transporters, channels, tran- role of protective protein (Zhou et al. 2014a). The induced scription factors, enzymes, phosphatases and so on (Chen et expression of genes involved in ROSs scavenging (CAT, al. 2015a). SOD, POD) and the increased activities of antioxidant en- The new technologies also provided us powerful tools zymes (CAT, SOD, POD) can remove excess ROS produced for identifications of genes, proteins and metabolites associ- by osmotic stress (Shi et al. 2014a, Ye et al. 2016). Up- ated with various stress tolerances (Table 2). We have se- regulation genes function as solute synthase (such as sucrose lected many candidate genes involved in different stresses

Table 2. Omics technologies used in bermudagrass stress study Type of Technology Main research result References stresses cadmium metabolomics thirty-nine metabolites included some amino acids, organic acids, sugars, and fatty acids are Xie et al. 2014 stress found to be responsive to Cd stress drought transcriptomics two hundred and seventy-seven drought responsive genes are identified, the up-regulated genes Zhou et al. 2014a stress contain genes related to drought avoidance and tolerance traits and stress signaling transcriptomics one hundred and twenty up-regulated genes are involved in proline biosynthesis, signal trans- Kim et al. 2009 duction pathways, protein repair systems, and removal of toxins, while 60 down-regulated genes are mostly related to basic plant metabolism proteomics stress-responsive proteins are mainly involved in metabolism, energy, cell growth/division, Zhao et al. 2011 protein synthesis and stress defense and there exists a different level of protein accumulation between hybrid and common bermudagrass proteomics thirty-nine proteins with significantly changed abundance between drought sensitive and Shi et al. 2014a tolerant variety were identified, most of which are involved in photosynthesis glycolysis, N-metabolism, tricarboxylicacid and redox pathways proteomics and about 75 proteins, mainly involved in photosynthesis, biodegradation of xenobiotics, oxidative Ye et al. 2015, 2016 metabolomics pentose phosphate, glycolysis and redox pathway are identified waterlog- proteomics and forty-five showed abundance changes after submergence treatment with 10 increased and 35 Ye et al. 2015 ging stress metabolomics decreased; 34 of 40 metabolites contents exhibited down-regulation or no significant changes cold stress transcriptomics photosynthesis, nitrogen metabolism and carbon fixation pathways play key roles in bermuda Chen et al. 2015a grass response to cold stress transcriptomics a total of 5867 genes are differentially expressed in cold acclimate versus non-acclimated Zhu et al. 2015 bermudagrass, large numbers of AP2, NAC and WRKY family members are associated with cold stress salt stress proteomics seventy-seven differentially expressed proteins are identified upon drought and salt stresses and Ye et al. 2016 most of which are known to be involved in glycolysis, oxidative pentose phosphate, photosynthesis and redox metabolic pathways transcriptomics it identifies candidate genes encoding TFs (MYB, bHLH, WRKY) involved in the regulation of Hu et al. 2015b lignin synthesis, reactive oxygen species (ROS) homeostasis controlled by peroxidases, and the regulation of phytohormone signaling that promote cell wall loosening and therefore root growth under salinity

8 Breeding Science Responses of bermudagrass to abiotic stresses Preview BS through the method, however, the function of the genes 1222–1230. need to be verified. The information presented in this review Bayrer, T.A. (2006) Wear tolerance of seeded and vegetatively propa- and the studies currently been conducted in laboratories gated bermuda grasses under simulated athletic traffic. [master’s worldwide should be very helpful when efficient stable thesis]. [Lexington (KY)]: University of Kentucky. transformation methods for bermudagrass become avail Beard, J.B. (1973) Turfgrass: Science and culture. Prentice-Hall, Englewood Cliffs, pp. 130. able. Our laboratory is now focusing on the improvement of Beard, J.B. (1989) Turfgrass water stress: Drought resistance compo- bermudagrass transformation technologies, including proto- nents, physiological mechanisms, and species-genotype diversity. plast transformation method (Huang et al. 2018). We can In: Takatoh, H. (ed.) Proc. of the Inter. Turf. Res. Conf., 6th, Tokyo. also express bermudagrass candidate genes in model plants 31 July–5 Aug. 1989. Japan. Soc. of Turf. Sci., Tokyo, Japan. like rice, tobacco and Arabidopsis, to identify their func- Bhardwaj, P., A.K. Chaturvedi and P. Prasad (2009) Effect of enhanced tions. Then we can improve the resistance of bermudagrass lead and cadmium in soil on physiological and biochemical attrib- to related adversity by transgenic means. At the same time, utes of Phaseolus vulgaris L. Nat. Sci. 7: 63–75. we can transfer resistance genes in bermudagrass to crops to Bizhani, S. and H. Salehi (2014) Physio-morphological and structural improve their resistance to related stress. As global climate changes in common bermudagrass and Kentucky bluegrass during change continues, it is essential for researchers to generate salt stress. Acta Physiol. Plant. 36: 777–786. more and new stress tolerant plants, including bermuda Bonos, S.A. and B.R. 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