Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol155.Issue1.2000:

Plant Science 155 2000 1 – 9 Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene Elumalai Sivamani a,1 , Ahmed Bahieldin b,1 , Jon M. Wraith c , Thamir Al-Niemi a , William E. Dyer a , Tuan-Hua David Ho d , Rongda Qu a, a Department of Plant Sciences, Montana State Uni6ersity, Bozeman, MT 59717 - 3140 , USA b Ain Shams Uni6ersity, Cairo, Egypt c Department of Land Resources and En6ironmental Sciences, Montana State Uni6ersity, Bozeman, MT 59717 - 3120 , USA d Department of Biology, Washington Uni6ersity, St Louis, MO 63130 , USA Received 21 May 1999; received in revised form 12 November 1999; accepted 16 November 1999 Abstract The ABA-responsive barley gene HVA1, a member of group 3 late embryogenesis abundant LEA protein genes, was introduced into spring wheat Triticum aesti6um L. cv. Hi-Line using the biolistic bombardment method. High levels of expression of the HVA1 gene, regulated by the maize ubi1 promoter, were observed in leaves and roots of independent transgenic wheat plants and were inherited by offspring generations. T 3 progenies of four selected transgenic wheat lines were tested under greenhouse conditions for tolerance of soil water deficit. Potted plants were grown under moderate water deficit and well-watered conditions, respectively. Two homozygous and one heterozygous transgenic lines expressing the HVA1 gene had significantly P B 0.01 higher water use efficiency values, 0.66 – 0.68 g kg − 1 , as compared to 0.57 and 0.53 g kg − 1 , respectively, for the non-expressing transgenic and non-transgenic controls under moderate water deficit conditions. The two homozygous transgenic plant lines also had significantly greater total dry mass, root fresh and dry weights, and shoot dry weight compared to the two controls under soil water deficit conditions. Results of this study indicate that growth characteristics were improved in transgenic wheat plants constitutively expressing the barley HVA1 gene in response to soil water deficit. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Barley HVA1 gene; Biomass productivity; LEA protein; Water deficit; Water use efficiency; Wheat transformation www.elsevier.comlocateplantsci

1. Introduction

Drought is an important environmental factor limiting the productivity of wheat and other crops world-wide. As irrigation water sources have be- come more scarce, development of crop cultivars with improved adaptation to drought is a major goal in many crop breeding programs. Of the physiological factors that affect drought tolerance in crops, the most important ones seem to be moderate water use through reduced leaf area and shorter growth duration, rooting ability to exploit deep soil moisture, the capacity for osmotic ad- justment, and control of non-stomatal water loss from leaves [24]. Drought tolerance breeding might be facilitated by marker-assisted selection in the near future based on the development of molecular linkage maps for crop species [24]. When breeding for drought tolerance, biomass productivity and water use efficiency WUE are Corresponding author. Present address: Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620. Tel.: + 1-919-515-2647; fax: + 1-919-515-7959. E-mail addresses : rongda –quncsu.edu R. Qu 1 Also author for correspondence. Present address: MAHYCO Life Sciences Research Centre, PO Box 76, Jalna, Maharashtra, India. Elumalai Sivamani and Ahmed Bahieldin are considered co-first authors. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 4 7 - 2 considered important agronomic characters [1]. There is increasing interest in improving WUE of crop cultivars so that plants can grow and yield well under water deficient conditions [2,12]. In addition to classical breeding approaches, recent work has used genetic transformation to introduce various genes into plants for better tolerance to water deficit. One strategy is to genetically engi- neer plants to accumulate compatible osmolytes, such as amino acids, sugars, or sugar alcohols, resulting in decreased osmotic potential and avoid- ance of water deficit. Mannitol [29], proline [19], fructan [25], and trehalose [15] have been shown to accumulate in transgenic tobacco plants when genes encoding the corresponding biosynthetic en- zymes were introduced, resulting in improved growth of transgenic plants under conditions of water deficit andor salt stress. Additional novel roles of these compounds in the protection of plants against water stress were proposed [3,15]. Another transgenic approach is to constitutively over-express certain plant proteins, such as late embryogenic abundant LEA proteins that accu- mulate during seed desiccation, and in vegetative tissues when plants experience water deficit. Groups of LEA proteins have been defined based on their amino acid sequence homologies [10]. Three LEA proteins have been demonstrated to play roles in water deficit tolerance. LE25, a group 4 LEA protein from tomato Lycopersicum escu- lentum L. improved resistance to high salinity and freezing when expressed in yeast Saccharomyces cere6isiae [18], while HVA1, a group 3 LEA protein from barley Hordeum 6ulgare L. con- ferred tolerance to soil water deficit and salt stress in transgenic rice plants [34]. Very recently, a wheat LEA protein, Em, was demonstrated to function as an osmoprotective molecule in yeast [27]. The HVA1 gene was first isolated from the aleurone layers of barley seeds as an ABA-in- ducible gene [16]. The deduced amino acid se- quence from the corresponding cDNA indicates that HVA1 encodes a 22 kDa protein which con- tains nine imperfect repeats of an 11 amino acid motif, a characteristic feature of group 3 LEA proteins. HVA1 mRNA was rapidly induced in young seedlings 3 days after imbibition by ABA and a series of stress conditions including partial dehydration, cold, NaCl, and heat. However, 7- day-old seedlings were much less responsive, indi- cating tight developmental control of HVA1 gene expression [17]. Accumulation of HVA1 protein in transgenic rice plants led to higher growth rates, delayed damage symptoms, and improved recov- ery upon removal of the stress conditions over non-transgenic controls when seedlings were sub- jected to water deficit and salt stress [34]. In tobacco, constitutive expression of the HVA1 gene resulted in delayed wilting and wilting at lower leaf water contents Ho, unpublished data. In this study, we investigated the performance of transgenic wheat lines constitutively expressing the HVA1 gene by measuring several agronomic characters under water deficient as well as under well-watered conditions. We found that three of four transgenic lines expressing the HVA1 gene yielded more biomass and used water more effi- ciently than the control under moderate water deficient conditions. The engineered plants per- formed similarly to non-transgenic plants under well-watered conditions. Our results indicate that constitutive expression of HVA1 gene helps im- prove wheat growth characteristics under water deficient conditions, and further demonstrate pos- sible applications of certain LEA protein genes in engineering crop plants for tolerance to water deficit.

2. Materials and methods