Plastid-Expressed Betaine Aldehyde Dehydrogenase Gene in Carrot Cultured Cells, Roots, and Leaves Confers Enhanced Salt Tolerance

Abstract

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50-to 54-fold more betaine (93-101 mmol g 21 dry weight of b-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mM NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mM), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.Salt stress is a major abiotic stress in plant agriculture. The problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the world's irrigated lands are affected by salinity (Zhu, 2001). Currently, high salinity limits crop production in 30% of the irrigated land in the United States and 20 million hectares globally. High salinity causes ion imbalance, toxic levels of cytoplasmic sodium, and drought stress (Ward et al., 2003). Plants utilize a number of protective mechanisms to maintain normal cellular metabolism and prevent damage to cellular components (Wood et al., 1996). One of the metabolic adaptations to salt stress is the accumulation of osmoprotectants. Gly betaine and b-Ala betaine are quaternary ammonium compounds that accumulate in many plant species in response to salt stress Rhodes and Hanson, 1993;Rathinasabapathi et al., 2001). Gly betaine protects the cell from salt stress by maintaining an osmotic balance with the environment (Robinson and Jones, 1986) and by stabilizing the quaternary structure of complex proteins (Papageorgiou and Murata, 1995). This substance occurs naturally in some crops, like sugar beet and cotton, as well as in many highly salt-or drought-tolerant wild plants, including halophytes (Rhodes and Hanson, 1993;Nishimura et al., 2001). However, many stress-suscep...

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

“…The relative expression level of transgenes in tomato fruit chromoplasts, however, was higher with other proteins (Ruf et al 2001) and in any case sufficient to induce significant metabolic changes (Wurbs et al 2007;Apel and Bock 2009). Very low levels of mRNA and GFP protein were detected in roots of transplastomic Nicotiana benthamiana plants (less than 2% of that in the leaves), albeit expression levels up to 40-75% of those in leaf chloroplasts could be achieved in petal leucoplasts of the same plants or in chromoplasts of carrot taproots (Kumar et al 2004a;Davarpanah et al 2009). …”

“…A full-length plastid rrn operon promoter, with both PEP and NEP consensus sequences, along with the ribosome-binding site of the phage T7 gene 10 leader and the 3 0 -UTR of the plastid gene encoding the ribosomal protein S16 were recently used to facilitate transgene expression in carrot chromoplasts (Kumar et al 2004a). A similar approach was adopted by Wurbs et al (2007), who used the atpI promoter containing both PEP and NEP signals, and the rps16 3 0 -UTR, to express carotenoidrelated genes in tomato fruits.…”

Plastid transformants of tomato selected using mutations affecting ribosome structure

“…Plastid transformation of tobacco (Nicotiana tabacum) is routine in a number of laboratories, but there are only a limited number of isolated reports relating to genera outside Nicotiana. These include tomato (Ruf et al 2001), rice (Khan and Maliga 1999), potato (Sidorov et al 1999), carrot (Kumar et al 2004) and several Brassicaceae, including Arabidopsis (Sikdar et al 1998), Brassica napus (Hou et al 2002) and Lesquerella fendleri (Skarjinskaia et al 2003). All of these investigators used biolistics to deliver DNA into chloroplasts.…”

Chloroplast Genetic Engineering

“…This provides yet another strategy for transgene containment. Chloroplast transgenic carrot plants have been engineered to withstand salt concentrations only halophytes could tolerate [16]. The Daniell laboratory has conferred several other plant traits, including herbicide [12], insect [17], and disease [18] resistance, drought [10], phytoremediation [19] and production of biopolymers [20].…”

“…These examples show that chloroplast also contains the machinery that allows for correct folding and disulfide bond formation, resulting in fully functional proteins. Finally, the transformation of non-green tissue plastids like cotton [14] and carrot [16] were recently achieved [6], further facilitating oral delivery of therapeutic proteins. To advance the concept of oral delivery of therapeutic proteins, an antibiotic free cp transformation system has been developed [27].…”