Biologia 68/5: 879—895, 2013Section Cellular and Molecular BiologyDOI: 10.2478/s11756-013-0229-8Glycine betaine biosynthesis in saltbushes (Atriplex spp.)under salinity stressShanthi Joseph 1 , Daniel Murphy 2 & Mrinal Bhave 1 **1 Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology,PO Box 218, Hawthorn, Victoria 3122, Australia; e-mail: mbhave@swin.edu.au2 Royal Botanic Gardens Melbourne, Private Bag 2000, Birdwood Avenue, South Yarra, Victoria 3141, AustraliaAbstract: Soil salinity and drought severely af fect all aspects of plant physiology, leading to signif icant losses of crop pro-ductivity and native biodiversity. A key to sustainable land use in such areas is to cultivate well-adapted native plants thatare also commercially important and have the appropriate gene pool. Glycine betaine (GB) is an osmoprotectant that im-parts salt and drought tolerance to some plants. It is also shown separately to provide signif icant health benef its to animalsand humans. We investigated whether Australian saltbushes, which are extremely salt and drought tolerant and also imparthealth benef its to grazing animals, may have the genetic basis for GB biosynthesis, explaining the two dif ferent observa-tions. Complementary DNAs encoding the two key enzymes of the plant GB biosynthesis pathway, choline monooxygenase(CMO) and betaine aldehyde dehydrogenase (BADH), were identif ied and analysed from Atriplex nummularia and Atriplexsemibaccata. The sequences showed the putative CMO proteins exhibited all functionally important features including theReiske-type cluster (2Fe-2S) and mononuclear non-heme Fe cluster, and the putative BADHs exhibited conservation ofactive site residues. The expression of both genes was found to be signif icantly up-regulated in leaf tissues under salt stress.The leaf tissues also showed accumulation of very high levels of GB, at 29.69 mmol/kg fresh weight for A. nummularia and42.68 mmol/kg fresh weight for A. semibaccata, which is several times higher than in cereal crops. The results demonstratea strong potential of cultivation of saltbushes for re-vegetation and as a perennial fodder in salinity and drought-af fectedareas.Key words: salinity; Atriplex; osmoprotectants; betaine aldehyde dehydrogenase; choline monooxygenase; biodiversity.Abbreviations: ALDH, aldehyde dehydrogenase; BADH, betaine aldehyde dehydrogenase; cDNA, complementary DNA;CMO, choline monooxygenase; cTP, chloroplast transit peptide; FW, fresh weight; GB, glycine betaine.IntroductionSalinity is a global problem that currently af fects about10 million hectares of arable land and has a signif icantnegative impact on agricultural productivity and sus-tainability, due to the salt-sensitive nature of most ce-real crops. Ef fective land management is thus criticalfor food security and biodiversity conservation. An idealstrategy is phytoremediation using well-adapted nativehalophytic plants, as these have diverse innate mech-anisms to combat salinity; some can also be used assources of food, fodder, ornamentals and/or chemicals.The Australian native saltbushes (Atriplex spp.)are extremely salt and drought tolerant (http://vro.dpi.vic.gov.au/dpi/vro/vrosite.nsf/pages/sss salinitywestern victoria). The seed germination of Atriplexprostrata and Atriplex patula (Katembe et al. 1998) andAtriplex centralasiatica (Liu et al. 2006) was not per-manently inhibited at high salt levels. Saltbushes of ferenvironmental benef its, such as reductions in soil ero-sion and surface salinity, groundwater recharge (Van-Bueren & Price 2004), excellent top cover due to hor-izontal growth (Harris et al. 2009), and an excep-tional choice for perennial re-vegetation and fodderdue to their long life span (over 60 years for somespecies). A unique Australian initiative, the KamarookaProject, undertaken by the Northern United ForestryGroup (NUFG, Bendigo, Australia; Agro-forestry andmixed perennials in a low rainfall saline landscape;http://www.daf f.gov.au/ data/assets/pdf f ile/0016/29122/vic agrofores try case study.pdf) has reclaimeda large salt-degraded area using selected native vegeta-tion that includes three saltbushes (Atriplex nummula-ria, Atriplex semibaccata, Atriplex amnicola; (http://www.daf f.gov.au/ data/assets/pdf f ile/0016/29122/vic agrofores try case study.pdf; P. Dyson & I. Rankin,personal communications, November 2009).The salinity and drought tolerance of saltbushes* Electronic supplementary material. The online version of this article (DOI: 10.2478/s11756-013-0229-8) contains supple-mentary material, which is available to authorized users.** Corresponding authorc ? 2013 Institute of Molecular Biology, Slovak Academy of Sciences
