Biological Functions of Antioxidants in Plant Transformation
By Dan, Yinghui
Abstract Browning and necrosis of transformed cells/ tissues, and difficulty to regenerate transgenic plants from the transformed cells/tissues (recalcitrance) are common in Agrobacterium-mediated transformation process in many plant species. In addition, most crop transformation methods that use NPTII selection produce a significant number of nontransgenic shoots, called “shoot escapes” even under stringent selection conditions. These common problems of plant transformation, (browning and necrosis of transformed cells/ tissues, recalcitrance, and the occurrence of shoot escapes) severely reduces transformation efficiency. Recent research indicates that reactive oxygen species (ROS) such as superoxide radical (O^sup -^^sub 2^), the hydrogen peroxide (H^sub 2^O^sub 2^), the hydroxyl radical (OH’, and the peroxyl radical (RO^sub 2^) may be playing an important role in tissue browning and necrosis during transformation. This review examines the role of ROS in in vitro recalcitrance and genetic transformation and the opportunities to improve transformation efficiency using antioxidants.
Keywords Agrobacterium * Reactive oxygen species * Oxidative burst * Necrosis
It is well known that transformation of plant genome using Agrobacterium tumefaciens is the exploitation of the process of pathogen infection. Normally, the initial response of plants to pathogen attacks is an oxidative burst with rapid and transient production of reactive oxygen species (ROS) (Wojtaszek 1997). This plant response indeed is a defense mechanism as ROS can kill the pathogenic bacteria or inhibit their growth (Wu et al. 1995). ROS production is usually followed by the hypersensitive response (HR) to pathogens leading to rapid cell death (necrosis) (Greenberg et al. 1994). Although the interaction between plant and Agrobacterium is not yet fully understood, several studies have reported necrosis and a poor survival rate of target plant tissues following Agrobacterium-mediated transformation (Perl et al. 1996; Enriquez- Obregon et al. 1997, 1998; Hansen 2000; Olhoft et al. 2001a, b; Chakrabarty et al. 2002; Das et al. 2002; Toldi et al. 2002; Dan et al. 2004; Zheng et al. 2005).
Many crop transformation methods that use NPTII selection have a common problem of regeneration of nontransgenic shoots while imposing kanamycin selection at the shoot formation stage. These nontransgenic shoots, referred to as “shoot escapes” that are different from “escapes” that we often refer as nontransgenic plants, occur in high frequencies in many crop species tested. For instance, the occurrence of ranging from 40% to 90% have been reported for apple (James et al. 1989), pear (Mourgues et al. 1996), banana (Murkute et al. 2003), grapevine (Perl et al. 1996), citrange (Moore et al. 1992; Pena et al. 1995a), sweet orange (Pena et al. 1995b; Cervera et al. 1998), and lime (Pena et al. 1997). In cauliflower Stipic et al. (2000), found that approximately 95% of shoots regenerated from selective media were shoot escapes. As with tissue necrosis, the exact reason for the occurrence of “shoot escapes” is unknown.
A different but related issue is the inability of plant tissues to respond to culture manipulations for the desired outcome (Benson 2000a). Under normal tissue culture conditions, even without transformation, many plant species fail to respond to culture manipulations (Benson 2000a). This phenomenon, referred to as “in vitro recalcitrance”, has been ascribed to several factors including cellular incompetence and necrosis but, again, the exact reason(s) for this problem remain unclear.
The three common problems described above, (1) browning/necrosis of transformed cells/tissues, (2) shoot escapes in transgenesis, and (3) in vitro recalcitrance, severely limit the number of transgenic plants that can be regenerated. Although seemingly unrelated, recent research is beginning to unravel a common factor underlying these problems, the production of ROS, which can cause growth inhibition, cell death, or alter plant metabolic pathways leading to poor regeneration of plants and the production of shoot escapes.
ROS include a number of chemically reactive molecules derived from oxygen whose functions have been reviewed (Fridovich 1989; Halliwell 1996, 1999; Betteridge 2000). Some of those molecules are extremely reactive, such as the hydroxyl radical, while some are less reactive (superoxide and hydrogen peroxide). Intracellular tree radicals, i.e., free, low molecular weight molecules with an unpaired electron, are often ROS and the two terms are, therefore, commonly used as equivalents. Free radicals and ROS can readily react with most biomolecules, starting a chain reaction of free radical formation. In order to stop this chain reaction, a newly formed radical must either react with another free radical or react with a free radical scavenger, such as an antioxidant.
ROS are formed and degraded by all aerobic organisms, leading to either physiological concentrations required for normal cell function or excessive quantities, the state called oxidative stress. Growing evidence has indicated that cellular reduction/oxidation (redox) status regulates various aspects of cellular function. Oxidative stress can elicit positive responses such as normal cellular proliferation, activation of transcription factors or gene expression, as well as negative responses such as growth inhibition or cell death (Palmer et al. 1987; Furchgott 1995; Sundaresan et al. 1995; Finkel 1998; Kamata and Hirata 1999; Patel et al. 1999; Rhee 1999). Excessive production of ROS often leads to oxidative stress, loss of cell function, and ultimately apoptosis or necrosis by its interference with various biomolecules, including proteins, lipids, and DNA (Marnett 2000). A balance between oxidant and antioxidant intracellular systems is hence vital for normal cell function, growth regulation, and adaptation to diverse growth conditions (Nordberg and Arner 2001).
Broadly, this review addresses some the biological aspects of ROS production and its manipulation to improve plant transformation. The emphasis will be on the causes and possible solutions to minimize regenerative recalcitrance due to cell death.
Antioxidant Definition and Actions
What is an antioxidant? An antioxidant by definition is a substance that significantly delays or prevents oxidation of its oxidizable substrate when present at low concentrations compared to those of its substrate (Halliwell and Gutteridge 1989; Halliwell 1990). The term “oxidizable substrate” includes almost everything found in living tissues, particularly proteins, lipids, carbohydrates, and DNA (Halliwell et al. 1995). Packer et al. (1995) stated that many criteria must be considered when evaluating the antioxidant potential of a compound. Some of these concerning chemical and biochemical aspects are: specificity of free radical quenching, metal chelating activity, interaction with other antioxidants, and effects on gene expression. Regarding preventive or therapeutic applications, other criteria, such as absorption and bioavailability, concentration in tissue/cell/extra-cellular fluid, and location (in aqueous or membrane domains or in both) are important.
The important ROS that cause damage to living cells and their production in vivo. Important ROS that cause damage to living cells are the superoxide radical (O^sup -^^sub 2^), the hydrogen peroxide (H^sub 2^O^sub 2^), the hydroxyl radical (OH’), and the peroxyl radical (RO2) (Halliwell et al. 1995).
Superoxide, which formed in vivo, is largely converted by superoxide dismutase (SOD)-catalyzed or nonenzymic dismutation into H^sub 2^O^sub 2^ (Fridovich 1989). Some enzymes, such as glycolate oxidase, also produce H^sub 2^O^sub 2^ directly in vivo (Chance et al. 1979; Halliwell and Gutteridge 1989). Unlike O^sup -^^sub 2^, H^sub 2^O^sub 2^ is able to cross biological membranes (Halliwell and Gutteridge 1989). Both O^sup -^^sub 2^ and H^sub 2^O^sub 2^ can find molecular targets to inflict direct damage, but their reactivity is limited (Halliwell et al. 1995). The molecular damage that can be done by O^sup -^^sub 2^ and H^sub 2^O^sub 2^ is considered to be due to their conversion into more reactive species, which have been reviewed by Halliwell and Gutteridge (1989, 1990).
The most important of the more reactive species is the hydroxyl radical (OH-’). Hydroxyl radical can be formed from O^sup -^^sub 2^ through at least four different mechanisms (Halliwell et al. 1995). One of the mechanisms requires traces of catalytic transition metal ions, of which iron and copper seem likely to be the most important in vivo (Igene et al. 1979; Kanner et al. 1987; Ramanathan and Das 1993; Miller et al. 1994).
A second mechanism requires the exposure to ionizing radiation which causes a steady state low rate production of OH’ formation within cells and in food by splitting water (von Sonntag 1987). Food irradiation (Elias 1994) for sterilization or prevention of germination will generate increased levels of OH’. The other means of OH’ formation involved is the reaction of O^sup -^^sub 2^ with the free radical nitric oxide (NO’) and hypochlorous acid (HOCl), respectively. Peroxyl radicals (RO’^sub 2^) are formed in both lipid peroxidation (Halliwell and Gutteridge 1989) and nonlipid systems, such as proteins (Davies et al. 1993; Dean et al. 1993). Decomposition of peroxides by heating or by transition metal ion catalysis can generate both peroxyl and alkoxyl radicals (Halliwell et al. 1995). The sites and actions of antioxidants in living cells. Main antioxidant actions include scavenging ROS or free radicals, inhibiting the generation of ROS, and chelating metals, as well as their effects on cell signaling pathways and on gene expression (Halliwell et al. 1995; Soobratteea et al. 2005). However, it seems that antioxidants that interfere with the activity of OH’ do not by direct OH’ scavenging, but by scavenging or blocking the formation of its precursors (O^sup -^^sub 2^, H^sub 2^O^sub 2^, HOC1, ONOO’) and/or by binding the transition metal ions needed for OH’ formation from O^sup -^^sub 2^ and H^sub 2^O^sub 2^ (Halliwell et al. 1995). In addition, many lipid-soluble chain-breaking antioxidants can have prooxidant properties, which induce oxidative stress either through creating reactive oxygen species or inhibiting antioxidant systems, under certain circumstances in vitro, often because they can bind Fe(III) or Cu(II) ions and reduce them to Fe^sup 2+^Or Cu+ (Mukai et al. 1993).
Antioxidants act as scavengers of ROS, such as the peroxyl radical either in aqueous phase (e.g., with radicals from DNA, thiols, and proteins) or in hydrophobic phase (food lipid, membrane, lipoprotein interior) (Halliwell et al. 1995). For example, glutathione reacts rapidly with free radicals generating from an attack of OH’ on DNA, at rate constants of about 10^sup 7^-10^sup 8^ M^sup -1^ s^sup -1^ (von Sonntag 1987; Fahey 1988). Other antioxidants, e.g., scavengers of the peroxyl radical such as chain breaking antioxidants (propyl gallate and alpha-tocopherol) that are inhibitors of lipid peroxidation, could act hydrophobically in lipids, cell membranes, and interior structure of lipoproteins.
Major Problems and Antioxidants Used in Plant Tissue Culture and Transformation
Hyperhydricity. Hyperhydricity, a physiological disorder occurring in plant tissue cultures, is associated oxidative damage (Chakrabarty et al. 2006). Hyperhydricity results in a general decrease in concentrations of reduced and oxidized pyridine nucleotides, reflecting a reduction in metabolic activity (Chakrabarty et al. 2006). The activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, ascorbate peroxidase, and glutathione reductase, were higher in hyperhydric leaves than in normal leaves, indicating that hyperhydricity was associated with oxidative stress (Chakrabarty et al. 2006). Measurements of chlorophyll fluorescence provided evidence of oxidative damage to the photosynthetic machinery in the hyperhydric leaves because the photochemical efficiency of photosystem II, the effective quantum efficiency, and photochemical quenching were all lower in the hyperhydric leaves (Chakrabarty et al. 2006). Toth et al. (2004) reported that the inclusion of glutathione in a culture medium could suppress the hyperhydricity of calli, thereby promoting regeneration of plantlets in a desiccation-tolerant plant, Ramonda myconi.
Recalcitrance. In vitro recalcitrance of plants has been associated with ROS production. Higher levels of free radical activity were found in recalcitrant genotypes of potato and grape as well as in nonembryogenic calli (rice) and reduced embryogenic capacity calli (carrot) compared to responsive genotypes and embryogenic calli, respectively, (Benson et al., 1992; Benson and Roubelakis-Angelakis 1992, 1994; Bailey et al. 1994; Bremner et al. 1997; Deighton et al. 1997). The production of the free radicals might lead to an increase in lipid peroxidation and subsequently have a negative effect on the morphogenetic capacity of plant tissue cultures (Benson 2000b).
Production of H^sub 2^O^sub 2^ coincided with the emergence of meristemoids and the formation of bud primordia in strawberry morphogenic calli (Tian et al. 2003). High O^sup -^^sub 2^ level, low H^sub 2^O^sub 2^ level, and little or no SOD activity were detected in calli possessing low organogenesis capacity. Adding N,N- diethyldithiocarbonate, an SOD inhibitor, to the regeneration medium promoted O^sup -^^sub 2^ production, inhibited H^sub 2^O^sub 2^ production and decreased the regeneration percentage, whereas the exogenous addition of H^sub 2^O^sub 2^ slightly promoted the potential for regeneration of shoot buds. The results suggest that H^sub 2^O^sub 2^ is directly correlated with the morphogenetic process in strawberry callus (Tian et al. 2003). Types II and III strawberry calli, which showed higher regeneration capacity, had five- and ninefold higher contents of intracellular H^sub 2^O^sub 2^, and three- and fourfold higher contents of intracellular O^sup – ^^sub 2^ compared to type I callus, which exhibited a much lower regeneration capacity (Tian et al. 2004). Types II and III calli also showed much higher activities of antioxidant enzymes than type I calli. The results indicated that ROS might play a dual role in the regeneration of strawberry calli. On one hand, a certain level of a typical ROS may have a positive effect on strawberry regeneration. On the other hand, high levels of another ROS are inhibitory to the expression of totipotency in strawberry calli (Tian et al. 2004). Similar study found that H^sub 2^O^sub 2^ promoted somatic embryogenesis of gladiolus at 100 [mu]M but it inhibited shoot organogenesis (Dutta and Datta 2003).
Antioxidants and the control of tissue browning and necrosis in plant tissue culture. Numerous studies reported tissue browning/ necrosis leading to poor plant regeneration in vitro and successful use of antioxidants solving the problems in the tissue culture history (Table 1). Most of the studies were associated with dicotyledonary plant species; however, several antioxidants such as ascorbate, cysteine, dithiothreitol (DTT), glutathione, and tocopherol were also successfully used in monocotyledonary plant species (Table 1). Among the studies, four of them had been well documented. Ziv and Halevy (1983) reported that using an antioxidant, DTT, controlled oxidative browning of tissues during in vitro propagation of Strelitzia reginae. Terminal and axillary buds, which were treated with a solution containing 0.04% DTT by submersing the buds for 24 h and then culturing the buds on an agar medium with charcoal or by culturing the buds on paper bridges in a liquid medium with 0.04% DTT, were able to develop to shoots. Also the degree of oxidative browning of the shoot tip expiants was reduced from a rating of 4 (without DTT) to a rating of 1.5. Shoot tips of apple (Malus pumild) rootstock M.26 turned brown immediately after being excised from expiants during in vitro propagation. The browned shoot tips would neither proliferate nor develop into plantlets (Nomura et al. 1998). Glutathione (GSH) was applied before the propagation to prevent browning of the shoot tip explants (Nomura et al. 1998). Shoot development from the explants, which were treated with 0.1 mM GSH solution by dipping them in the solution prior to culture, was compared with that of the explants without GSH treatment (control). In the GSH treatment, 100% of shoot tips developed into normal shoots after 120 d, whereas the result from the control was 40%. The results showed that application of GSH prior to the culture promoted the normal development of shoot tips. Apparently the major effect of GSH on the shoot tip development was the protection from browning of the shoot tips. For in vitro propagation of Protea cynaroides, oxidative browning of shoot segment expiants is a major problem (Wu and Toit 2004). However, almost all expiants, which were immersed in a solution containing 100 mg/l ascorbic acid and 1,500 mg/l citric acid for 1 h, and grown under 16 h photoperiod, had a 100% of the expiants survived and developed shoots, while only 20% of the expiants survived without the antioxidant treatment (Wu and Toit 2004). Adonis amurensis is a perennial ornamental plant whose shoot tip expiant darkening is a major obstacle to establish in vitro propagation (Park et al. 2006). Normally, about 20% of the expiants survive the initial stages of the culture (Park et al. 2006). However, when they were soaked in an antioxidant solution containing 300 mg/l ascorbic acid and 300 mg/l citric acid for 30 min prior to the culture, survival rate was about two times higher (53.3%) than the nontreated control (23.0%) (Park et al. 2006).
Here, I have classified the major antioxidants used in plant tissue culture into three groups based on their in vitro functions. The first group of antioxidants can both reduce tissue browning and promote organogenesis, somatic embryogenesis, and shoot growth from buds during micropropagation across different plant species (Table 1). These antioxidants are ascorbic acid, citric acid, DTT, polyvinyl-pyrrolidone (PVPP), and vitamin C (Table 1). The second group of antioxidants can enhance shoot, root, and plant growth in different plant species. They are cysteine, phenoxane, 3-ter-butyl- 4-hydroxyanisole, and vitamin E (Table 1). The third group of antioxidants not only can promote callus and shoot organogenesis but also inhibit somatic embryogenesis (Table 1). These antioxidants are ascorbate, glutathione, and alpha-tocopherol (Table 1). In addition, glutathione in culture medium could suppress the hyperhydricity of calli as shown in the desiccationtolerant plant, R. myconi (Toth et al. 2004).
Antioxidants and the control of tissue browning and necrosis in plant transformation. Since 1996, tissue browning/necrosis associated with Agrobaterium-mediated transformation have been reported in different types of explants of both dicotyledonous and monocotyledonous species. The examples include the browning/ necrosis occurred in embryogenic calli and leaf disks of grape (Pu and Goodman 1992; Perl et al. 1996; Das et al. 2002), cotyledonary nodes of soybean (Olhoft et al. 200la, b), hypocotyls of cauliflower (Chakrabarty et al. 2002), leaf segments of C. plantagineum (Toldi et al. 2002), epicotyl sections of peanut (Zheng et al. 2005), and cotyledons of tomato (Dan et al. 2004), leaf spindle sections of sugarcane (Enriquez-Obregon et al. 1997, 1998; Gustavo et al. 1998), shoot meristem sections, and calli of rice (Enriquez-Obregon et al. 1999), and suspension cells, immature embryos, and embryogenic calli of corn (Hansen 2000). From these reports, it seems that dicotyledonous species are more susceptible to tissue browning/ necrosis than monocotyledonous plants, but it can be controlled by the use of antioxidants PVPP, DTT, ascorbic acid, cysteine, glutathione, selenite, tocopherol, or lipoic acid (Table 2). It appears that ascorbic acid and cysteine are suitable to minimize browning/necrosis for both dicotyletonary and monocotyledonary plant species, but the rest of the antioxidants was largely experimented with dicotyletonous plants. Table 1. Antioxidants used in plant tissue culture
Perl et al. (1996) found that short exposures of embryogenic calli of Vitis vinifera cv. Superior seedless to a diluted solution of Agrobacterium resulted in tissue necrosis. The necrosis seemed to be oxygen-dependent and correlated with elevated levels of peroxides. Therefore, the inclusion of both 1% PVPP and 2 mg/l DTT in coculture medium was found to improve expiant viability during and after cocultivation. They observed that the necrosis of the embryogenic calli was completely inhibited by these antioxidants while Agrobacterium virulence was not affected. These antioxidants enabled the recovery of stable transgenic grape plants resistant to hygromycin. In another study Das et al. (2002) successfully used 1% PVPP and 2 mg/l DTT to control browning and necrosis in grape leaf tissue expiants in a coculture medium during Agrobacterium-mediated transformation.
Table 2. Antioxidants used in plant transformation
It was found that the coculture of sugarcane leaf spindle sections with A. tumefaciens induced a rapid necrosis of the tissue (Enriquez-Obregon et al. 1997, 1998; Gustave et al. 1998). To minimize the necrosis, the leaf spindle expiants were incubated in a liquid medium containing 15 mg/1 ascorbic acid, 40 mg/1 cysteine, and 2 mg/1 silver nitrate for 60 h in the dark prior to inoculation with Agrobacterium and then cocultured in a cocultivation medium with the same antioxidants at the same concentrations, respectively, after the inoculation with Agrobacterium. By doing so, the percentage of the expiant viability was increased from 10% (without the antioxidants) to 90%. In addition, the percentage of GUS positive expiants was increased from 0% (without the antioxidants) to 100%. Enriquez-Obregon et al. (1999) also investigated the effects of the three compounds on the necrosis of shoot meristem expiants prior to infection in rice transformation. The expiants, which were incubated in a liquid medium containing 20 mg/1 ascorbic acid, 40 mg/1 cysteine, and 5 mg/1 silver nitrate for 6 h in the dark, had an average of 6% of the each expiant area producing the necrosis, but the explants without the antioxidant treatment had 80% of the each expiant area producing the necrosis. The antioxidant treatment increased rice transformation efficiency from 17% without the antioxidant treatment to 30%. Similarly, Olhoft et al. (2001a, b) increased Agrobacterium infection from 37% (without cysteine) to 91% in the soybean cotyledonary node region by including 400 mg/l cysteine in cocultivation medium, subsequently, resulting in a twofold increase in transformation efficiency (2.1% with cysteine vs. 0.9% without cysteine). Browning/necrosis on the expiant tissues was also reduced. The studies also showed that the frequency of transformed cells was increased only when cysteine was present during cocultivation of Agrobacterium and cotyledonary-node explants. Later, the authors reported the cocultivation medium with 3.3 mM cysteine, plus 1 mM DTT resulted a significant higher transformation efficiency (12.7%) than that either with cysteine alone (7.7%) or no cysteine (0.7%) when using hygromycin B selection (Olhoft et al. 2003). Applying 400 mg/1 cysteine in the coculture medium increased both the frequency of transient a-glucuronidase (GUS) expression in target cells of com (56% with cysteine vs. 17% without cysteine) and the stable transformation frequency (6.2% with cysteine vs. 0.2% without cysteine) (Frame et al. 2002). However, cysteine reduced the percentage of the immature zygotic embryos giving rise to embryogenic Type II callus from 99% when noninfected immature zygotic embryos incubated on cocultivation medium without cysteine to 52% when the embryos cultured on cocultivation medium containing cysteine (Frame et al. 2002). Zeng et al. (2004) further confirmed that the inclusion of 400 mg/l cysteine in a cocultivation medium increased stable transformation from 0.2% (without cysteine) to 5.9% in soybean Agrobacterium-mediated transformation.
Adding glutathione to the selection medium reduced hyperhydricity of leaf expiants, increased leaf expiant viability, and increased the frequency of transformation from 13% (without glutathione) to 45% in Agrobacteriummediated transformation of a desiccation- tolerant plant, Craterostigma plantagineum (Toldi et al. 2002). Zheng et al. (2005) investigated the effects of antioxidants, including ascorbic acid, sodium selenite, DL-a-tocopherol, and glutathione in a cocultivation medium, on ROS production, antioxidant activity, and stable transformation efficiency during peanut Agrobacterium-mediated transformation. They found that glutathione, tocopherol, and selenite not only eliminated the formation of H^sub 2^O^sub 2^ produced in wound tissue during preparation of leaf expiants and their cocultivation with A. tumefaciens, but also decreased malondialdehyde (MDA) formation and enhanced the activities of the antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). The inclusion of 100 mg/l glutathione, 50 mg/l tocopherol, and 20 mg/l selenite in the cocultivation medium increased the transformation frequencies from 3.9% (no antioxidant) to 14.6%, 10.3%, and 12.4%, respectively (Zheng et al. 2005).
Lipoic acid (LA) is a sulfur-containing compound involved in several multienzyme complexes such as pyruvate dehydrogenase, ct- ketoglutarate dehydrogenase, branched-chain keto acid dehydrogenase, and glycine decarboxylase complex (Packer et al. 1995). In animals, free LA and dihydrolipoic acid are metabolic antioxidants that are able to scavenge most reactive oxygen species, to recycle other antioxidants such as vitamin C, glutathione, and vitamin B, and to increase the expression of genes involved in the regulation of normal growth and metabolism as well as redox regulation of gene transcription (Packer et al. 1995; Packer and Tritschler 1996; Packer et al. 1997). Therefore, LA was investigated in Agrobacteriummediated transformation across five different plant species, and it has significantly improved the transformation methods, even for recalcitrant genotypes (Dan et al. 2004; Dan 2006). Frequencies of independent transgenic plant events were increased in soybean from 0.6% to 3.6%, in potato from 3% to 19%, in tomato from 28% to 94%, and in wheat from 2.9% to 5.4%. The frequency of putative transgenic embryo was increased in cotton from 41% to 61%. The frequencies of shoot escapes were reduced in soybean from 92% to 72%, in potato from 50% to 16%, and in tomato from 91% to 53%, under the optimal conditions. This study also demonstrated that an increase of the transformation frequency and reduction of escapes in tomato were accompanied by a twofold reduction in the severity of the browning/necrosis of Agrobacterium-toansfomied tissues, a twofold increase in the survivability of the transformed tissues, a fourfold increase in the percentage of transgenic shoots, and a threefold reduction of the percentage of shoot escapes when using LA under optimal conditions. The application of LA in plant transformation has dramatically solved the three common problems in plant transformation: browning/necrosis of the transformed tissues, recalcitrance, and shoot escapes, which severely limit the number of transgenic plants produced.
Analysis of published information indicates that the antioxidants used in plant transformation can be classified into two groups based on their biological function. The first group, consisting of ascorbic acid, cysteine, DTT, lipoic acid, and PVPP, functions to reduce expiant necrosis, increase viability of expiants, and improve transformation efficiency while the other one, which include glutathione, selenite, and alpha-tocopherol, reduces hyperhydricity and ROS and increases transformation efficiency (Table 2).
Programmed CeU Death Associated with ROS in Plant
Mechanism of plant cell death induced by Agrobacterium. Hansen (2000) reported that the cell death triggered by Agrobacterium spp. in maize tissues had several features of apoptosis, which were DNA fragmentation, and cytological changes, and cytochrome c release. She further showed the two antiapoptotic genes from baculovirus, p35, and lap had the ability to prevent the onset of the apoptosis in maize tissues. p35 is reported to act as a direct inhibitor of caspases whereas lap may act upstream to prevent activation of caspases. Caspases control most events of apoptosis in vertebrates and in invertebrates and are responsible, either directly or indirectly, for the cleavage of cellular proteins. The proteins include nuclear proteins such as poly (ADP-ribose) polymerase, DNA- dependent protein kinase, and lamins, as well as actin (Nagata 1997). The evidence that these genes could affect the reaction of maize cells to an apoptotic stimulus strongly indicates that plant may have caspase-like proteases regulating apoptosis. A caspase- like proteolytic activity was also reported in tobacco plants undergoing an HR triggered by an infection with Tobacco mosaic virus (del Pozo and Lam 1998). Molecular identification of ROS signaling during programmed cell death (PCD) in plant. One of the first genes identified and isolated in ROS-associated plant cell death is LSDl that encodes a zinc-finger protein. This protein, together with two other zinc-finger proteins (LOLl and LOL2), could act as a molecular rheostat, sensing changes in ROS homeostasis, thereby controlling apoptosis through the regulation of apoptotic genes (Dietrich et al. 1997; Epple et al. 2003). The inactivation of the Arabidopsis Executerl gene completely abolished singlet oxygeninduced cell death (Wagner et al. 2004). The chloroplastic protein, which is encoded by Executerl, might perceive nonscavenged singlet oxygen species within the chloroplast. Other important stress signal transducers include mitogen-activated protein kinases (MAPKs) that act upstream of the oxidative burst during ozone treatment and the HR (Ren et al. 2002; Samuel and Ellis 2002). The primary ROS activated tobacco MAPK is the salicylic acidinduced protein kinase, which is required during harpindependent PCD (Samuel et al. 2005). A MAPKKK of alfalfe activates cell death induced by H^sub 2^O^sub 2^ through a specific MAPK-scaffolding action (Nakagami et al. 2004). A recent finding demonstrates the conserved Arabidopsis BCL2associated athanogene protein has been shown to be induced by H^sub 2^O^sub 2^ and capable of provoking PCD in both yeast (Saccharomyces cerevisiae) and plants (Kang et al. 2006).
Mechanism to control programmed cell death associated with ROS in plant. Several recent studies have begun to unravel the possible regulatory mechanisms of programmed cell death associated with ROS in plants (Breusegem and Dat 2006; Patel et al. 2006). The most extensively studied form of plant PCD is HR to pathogen infection (Greenberg and Yao 2004; Soosaar et al. 2005). Recent evidence indicates that autophagy, which is induced during the plant defense response, is one mechanism by which HR-PCD is controlled (Liu et al. 2005). Autophagy is a process hi which cytosol and organelles are sequestered within doublemembrane vesicles that deliver the contents to the lysosome/vacuole for degradation and recycling of the resulting macromolecules (Klionsky 2005). Several AuTophaGy (ATG) genes, which are required for autophagic activity, have identified via genetic screens in yeast (Levine and Klionsky 2004). The findings of Liu et al. (2005) imply that there is a prodeath signal that moves out of the HR lesion into the surrounding uninfected tissue. Therefore, autophagy most likely alters the induction, movement and/or recognition of the pro-PCD signal. Autophagy is not required for HR-PCD execution but is required to limit PCD to the infection sites in plant and might prevent a prodeath signal from initiating PCD in healthy tissue (Liu et al. 2005). Another study also suggests that autophagy has an anti-PCD function in innate immunity (Patel et al. 2006). However, examination of the function of ATG genes in other higher eukaryotes indicates that autophagy might have a dual role: pro- and/or anti-cell death (Yu et al. 2004; Boya et al. 2005; Pattingre et al. 2005). The study from Torres and Dangl (2005) suggest that ROS and nitric oxide are possible candidates for a pro-PCD signal. As extracellular ROS are produced before the onset of HR-PCD, ROS have been thought to function as pro- PCD signals, either by directly killing the pathogen or by acting as signaling molecules that mediate defense responses (Torres and Dangl 2005).
Apoptosis functions as an important defense strategy by host cells against viral invasion. Many viruses contain the antiapoptotic genes to block the defense-by-death response of host cells (Wang et al. 2004). The expression of antiapoptotic genes such as iap and p35 from baculoviruses, ced-9 from Caenorhabditis elegans and bcl-2 from humans in tobacco, tomato, and passion fruit plant (Passiflora spp) suppressed the extensive cell death caused by fungal and bacterial pathogens and also enhanced resistance to some abiotic stresses such as wounding, salt, cold, UV, and herbicides (Dickman et al. 2001; Lincoln et al. 2002; Chen and Dickman 2004; Li and Dickman 2004; Xu et al. 2004; Freitas et al. 2007). Particularly, baculovirus p35 and iap genes were expressed in com embryos and embryogenic calli and their expression reduced tissue browning 3 d after cocultivation with A. tumefaciens (Hansen 2000). These antiapoptotic genes can be engineered to be expressed in plants for controlling tissue browning/ necrosis and enhancing plant transformation efficiency, especially in recalcitrant or poorly transformable plant species.
Potential Mechanisms of Antioxidants for Improving Plant Transformation
Exposure of plant tissues to Agrobacterium during plant transformation leads to browning/necrosis of targeted cells/ tissues, which affect transformation efficiency (Kuta and Tripathi 2005). Browning/necrosis of targeted cells/tissues affects plant transformation in two ways. Browning/ necrosis may occur in transformed cells within expiant tissues, inhibiting regeneration of the transformed cells/ tissues. secondly, necrotic tissues are known to accumulate antimicrobial substances (Goodman and Novacky 1994), which may inhibit the potential of Agrobacterium to colonize plant cells and transfer T-DNA. Thirdly, the active release of chemical signals, which induce the vir genes in Agrobacterium, occurs only in living cells (Shaw et al. 1991). This also could reduce the potential of Agrobacterium to transfer T-DNA into plants. The inhibition of regeneration of transformed cells/tissues may promote growth of non-transformed cells/tissues even under selective pressures and, subsequently, result in production of shoot escapes. With regard to Agrobaterium, the ROS, produced during attempted transfection could be toxic enough to directly kill the attacking Agrobacterium (Wojtaszek 1997), thereby subsequently preventing Agrobacterium from colonizing plant cells and transferring T-DNA into plants.
Perl et al. (1996) observed that elevated levels of peroxidase activity in grape tissues correlated with Agrobacterium-induced necrosis in the host during Agrobacterium-mediated transformation. Peroxidase is known to mediate oxidative cross-linking of structural proteins in the cell wall (Somssich and Hahlbrock 1998), and the Agrobacterium-induced increase in peroxidase activity in grape tissues could further confirm the role of oxidative burst in HR in plants to Agrobacterium infection. Deng et al. (1995) demonstrated that at least two genes residing within the T-DNA region of Agrobacterium are responsible for inducing necrosis in grape tissues. Furthermore, the aviR gene in Agrobacterium vitis was found to be associated with Agrobacterium-induced HR (Zheng et al. 2003). aviR is homologous to luxR, which implies that the ^groeactentww- induced HR is regulated by a quorum-sensing mechanism. The Agrobacterium-mdaced HR could lead to rapid and large generation of ROS in target plant cells, resulting in plant cells/tissues necrosis, oxidative stress on the invading Agrobacterium cells, production of toxic antibacterial substances, and the deleterious effects on DNA molecules, especially at the site of oxidative burst (Zheng et al. 2003). All of these factors could significantly reduce the efficiency of stable transformation of plants.
Preliminary research on Agrobaterium-mduced plant cell death has shown that Agrobacterium likely causes browning/necrosis of transformed plant cells/tissues in vitro and ROS production during Agrobacterium infection in vivo induces necrosis. The ROS could kill the attacking Agrobacterium, thereby preventing Agrobacterium from infecting plant cells/tissues and delivering T-DNA into plants. Also, the necrosis prevents regeneration of transformed cells/ tissues.
Using antioxidants in plant tissue culture and transformation reduces the browning/necrosis of nontransformed and transformed cells/tissues and the frequencies of shoot escapes. In addition, the antioxidants increase the stable transformation efficiencies across dicotyledonary and monocotyledonay plant species, indicating their potential roles of controlling ROS for efficient production of transgenic plants.
Advancing Agrobacterium-mediated transformation technology requires an understanding of the mechanisms of Agrobacterium- induced browning/necrosis and the roles of antioxidants in facilitating plant transformation. The functions of the antioxidants described above have raised important questions. Do ROS produce when Agrobacterium infection takes place during in vitro transformation? Do the ROS cause the browning/necrosis of transformed cells/ tissues? Do the antioxidants scavenge and/or down-regulate the ROS through regulating the expression of genes involving ROS-generating systems such as NADPH oxidase or pH-dependent cell wall peroxidase? Do the ROS scavenging and/or down-regulating activities prevent the browning/necrosis of transformed cells/tissues? Or do the antioxidants alter the expression of genes that play an important role in the regeneration of transformed cells/ tissues by regulating the production of ROS that damage cells/tissues, or by activating other metabolic pathways? Once these questions are addressed, we can control apoptotic responses during wounding and Agrobacterium infection in plant transformation more effectively. This will undoubtedly lead to the development of more efficient plant transformation systems.
Acknowledgments The author thanks Dr. Richard E. Veilleux and Ms. Helen J. Hodges for English editing of the manuscript.
Received: 21 July 2006 /Accepted: 4 February 2008 / Published online: 18 March 2008 / Editor: P. Lakshmanan (c) The Society for In Vitro Biology 2008
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Y. Dan (*)
Institute for Sustainable and Renewable Resources,
Institute for Advanced Learning and Research,
150 Slayton Avenue,
Danville, VA 24540, USA
Department of Horticulture and Forestry,
Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061, USA
Copyright Society for In Vitro Biology May/Jun 2008
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