Biological functions of antioxidants in...

Tag : zinc selenite
Normally, the initial response of plants to pathogen attacks is anoxidative burst with rapid and transient production of reactiveoxygen species (ROS) (Wojtaszek 1997). This plant response indeedis a defense mechanism as ROS can kill the pathogenic bacteria orinhibit their growth (Wu et al. 1995). ROS production is usuallyfollowed by the hypersensitive response (HR) to pathogens leadingto rapid cell death (necrosis) (Greenberg et al. 1994). Althoughthe interaction between plant and Agrobacterium is not yet fullyunderstood, several studies have reported necrosis and a poorsurvival rate of target plant tissues followingAgrobacterium-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 etal. 2004; Zheng et al. 2005).

Many crop transformation methods that use NPTII selection have acommon problem of regeneration of nontransgenic shoots whileimposing kanamycin selection at the shoot formation stage. Thesenontransgenic shoots, referred to as "shoot escapes" that aredifferent from "escapes" that we often refer as nontransgenicplants, occur in high frequencies in many crop species tested. Forinstance, the occurrence of ranging from 40% to 90% have beenreported 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 (Penaet al. 1995b; Cervera et al. 1998), and lime (Pena et al. 1997). Incauliflower Stipic et al. (2000), found that approximately 95% ofshoots regenerated from selective media were shoot escapes. As withtissue necrosis, the exact reason for the occurrence of "shootescapes" is unknown.

A different but related issue is the inability of plant tissues torespond to culture manipulations for the desired outcome (Benson2000a). Under normal tissue culture conditions, even withouttransformation, many plant species fail to respond to culturemanipulations (Benson 2000a). This phenomenon, referred to as "invitro recalcitrance", has been ascribed to several factorsincluding cellular incompetence and necrosis but, again, the exactreason(s) for this problem remain unclear.

The three common problems described above, (1) browning/necrosis oftransformed cells/tissues, (2) shoot escapes in transgenesis, and(3) in vitro recalcitrance, severely limit the number of transgenicplants that can be regenerated. Although seemingly unrelated,recent research is beginning to unravel a common factor underlyingthese problems, the production of ROS, which can cause growthinhibition, cell death, or alter plant metabolic pathways leadingto poor regeneration of plants and the production of shoot escapes.

ROS include a number of chemically reactive molecules derived fromoxygen whose functions have been reviewed (Fridovich 1989;Halliwell 1996, 1999; Betteridge 2000). Some of those molecules areextremely reactive, such as the hydroxyl radical, while some areless reactive (superoxide and hydrogen peroxide). Intracellulartree radicals, i.e., free, low molecular weight molecules with anunpaired electron, are often ROS and the two terms are, therefore,commonly used as equivalents. Free radicals and ROS can readilyreact with most biomolecules, starting a chain reaction of freeradical formation. In order to stop this chain reaction, a newlyformed radical must either react with another free radical or reactwith a free radical scavenger, such as an antioxidant.

ROS are formed and degraded by all aerobic organisms, leading toeither physiological concentrations required for normal cellfunction or excessive quantities, the state called oxidativestress. Growing evidence has indicated that cellularreduction/oxidation (redox) status regulates various aspects ofcellular function. Oxidative stress can elicit positive responsessuch as normal cellular proliferation, activation of transcriptionfactors or gene expression, as well as negative responses such asgrowth inhibition or cell death (Palmer et al. 1987; Furchgott1995; Sundaresan et al. 1995; Finkel 1998; Kamata and Hirata 1999;Patel et al. 1999; Rhee 1999). Excessive production of ROS oftenleads to oxidative stress, loss of cell function, and ultimatelyapoptosis or necrosis by its interference with variousbiomolecules, including proteins, lipids, and DNA (Marnett 2000). Abalance between oxidant and antioxidant intracellular systems ishence vital for normal cell function, growth regulation, andadaptation to diverse growth conditions (Nordberg and Arner 2001).

Broadly, this review addresses some the biological aspects of ROSproduction and its manipulation to improve plant transformation.The emphasis will be on the causes and possible solutions tominimize regenerative recalcitrance due to cell death.

Antioxidant Definition and Actions

What is an antioxidant? An antioxidant by definition is a substancethat significantly delays or prevents oxidation of its oxidizablesubstrate when present at low concentrations compared to those ofits substrate (Halliwell and Gutteridge 1989; Halliwell 1990). Theterm "oxidizable substrate" includes almost everything found inliving tissues, particularly proteins, lipids, carbohydrates, andDNA (Halliwell et al. 1995). Packer et al. (1995) stated that manycriteria must be considered when evaluating the antioxidantpotential of a compound. Some of these concerning chemical andbiochemical aspects are: specificity of free radical quenching,metal chelating activity, interaction with other antioxidants, andeffects on gene expression. Regarding preventive or therapeuticapplications, other criteria, such as absorption andbioavailability, concentration in tissue/cell/extra-cellular fluid,and location (in aqueous or membrane domains or in both) areimportant.

The important ROS that cause damage to living cells and theirproduction in vivo. Important ROS that cause damage to living cellsare the superoxide radical (O^sup -^^sub 2^), the hydrogen peroxide(H^sub 2^O^sub 2^), the hydroxyl radical (OH'), and the peroxylradical (RO2) (Halliwell et al. 1995).

Superoxide, which formed in vivo, is largely converted bysuperoxide dismutase (SOD)-catalyzed or nonenzymic dismutation intoH^sub 2^O^sub 2^ (Fridovich 1989). Some enzymes, such as glycolateoxidase, also produce H^sub 2^O^sub 2^ directly in vivo (Chance etal. 1979; Halliwell and Gutteridge 1989). Unlike O^sup -^^sub 2^,H^sub 2^O^sub 2^ is able to cross biological membranes (Halliwelland Gutteridge 1989). Both O^sup -^^sub 2^ and H^sub 2^O^sub 2^ canfind molecular targets to inflict direct damage, but theirreactivity is limited (Halliwell et al. 1995). The molecular damagethat can be done by O^sup -^^sub 2^ and H^sub 2^O^sub 2^ isconsidered to be due to their conversion into more reactivespecies, which have been reviewed by Halliwell and Gutteridge(1989, 1990).

The most important of the more reactive species is the hydroxylradical (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 metalions, of which iron and copper seem likely to be the most importantin vivo (Igene et al. 1979; Kanner et al. 1987; Ramanathan and Das1993; Miller et al. 1994).

A second mechanism requires the exposure to ionizing radiationwhich causes a steady state low rate production of OH' formationwithin cells and in food by splitting water (von Sonntag 1987).Food irradiation (Elias 1994) for sterilization or prevention ofgermination will generate increased levels of OH'. The other meansof OH' formation involved is the reaction of O^sup -^^sub 2^ withthe free radical nitric oxide (NO') and hypochlorous acid (HOCl),respectively. Peroxyl radicals (RO'^sub 2^) are formed in bothlipid peroxidation (Halliwell and Gutteridge 1989) and nonlipidsystems, such as proteins (Davies et al. 1993; Dean et al. 1993).Decomposition of peroxides by heating or by transition metal ioncatalysis can generate both peroxyl and alkoxyl radicals (Halliwellet al. 1995). The sites and actions of antioxidants in livingcells. Main antioxidant actions include scavenging ROS or freeradicals, inhibiting the generation of ROS, and chelating metals,as well as their effects on cell signaling pathways and on geneexpression (Halliwell et al. 1995; Soobratteea et al. 2005).However, it seems that antioxidants that interfere with theactivity of OH' do not by direct OH' scavenging, but by scavengingor blocking the formation of its precursors (O^sup -^^sub 2^, H^sub2^O^sub 2^, HOC1, ONOO') and/or by binding the transition metalions needed for OH' formation from O^sup -^^sub 2^ and H^sub2^O^sub 2^ (Halliwell et al. 1995). In addition, many lipid-solublechain-breaking antioxidants can have prooxidant properties, whichinduce oxidative stress either through creating reactive oxygenspecies or inhibiting antioxidant systems, under certaincircumstances in vitro, often because they can bind Fe(III) orCu(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 radicaleither in aqueous phase (e.g., with radicals from DNA, thiols, andproteins) or in hydrophobic phase (food lipid, membrane,lipoprotein interior) (Halliwell et al. 1995). For example,glutathione reacts rapidly with free radicals generating from anattack of OH' on DNA, at rate constants of about 10^sup 7^-10^sup8^ M^sup -1^ s^sup -1^ (von Sonntag 1987; Fahey 1988). Otherantioxidants, e.g., scavengers of the peroxyl radical such as chainbreaking antioxidants (propyl gallate and alpha-tocopherol) thatare inhibitors of lipid peroxidation, could act hydrophobically inlipids, cell membranes, and interior structure of lipoproteins.

Major Problems and Antioxidants Used in Plant Tissue Culture andTransformation

Hyperhydricity. Hyperhydricity, a physiological disorder occurringin plant tissue cultures, is associated oxidative damage(Chakrabarty et al. 2006). Hyperhydricity results in a generaldecrease in concentrations of reduced and oxidized pyridinenucleotides, 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 thanin normal leaves, indicating that hyperhydricity was associatedwith oxidative stress (Chakrabarty et al. 2006). Measurements ofchlorophyll fluorescence provided evidence of oxidative damage tothe photosynthetic machinery in the hyperhydric leaves because thephotochemical efficiency of photosystem II, the effective quantumefficiency, and photochemical quenching were all lower in thehyperhydric leaves (Chakrabarty et al. 2006). Toth et al. (2004)reported that the inclusion of glutathione in a culture mediumcould suppress the hyperhydricity of calli, thereby promotingregeneration of plantlets in a desiccation-tolerant plant, Ramondamyconi.

Recalcitrance. In vitro recalcitrance of plants has been associatedwith ROS production. Higher levels of free radical activity werefound in recalcitrant genotypes of potato and grape as well as innonembryogenic calli (rice) and reduced embryogenic capacity calli(carrot) compared to responsive genotypes and embryogenic calli,respectively, (Benson et al., 1992; Benson and Roubelakis-Angelakis1992, 1994; Bailey et al. 1994; Bremner et al. 1997; Deighton etal. 1997). The production of the free radicals might lead to anincrease in lipid peroxidation and subsequently have a negativeeffect on the morphogenetic capacity of plant tissue cultures(Benson 2000b).

Production of H^sub 2^O^sub 2^ coincided with the emergence ofmeristemoids and the formation of bud primordia in strawberrymorphogenic 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 weredetected in calli possessing low organogenesis capacity. AddingN,N- diethyldithiocarbonate, an SOD inhibitor, to the regenerationmedium promoted O^sup -^^sub 2^ production, inhibited H^sub 2^O^sub2^ production and decreased the regeneration percentage, whereasthe exogenous addition of H^sub 2^O^sub 2^ slightly promoted thepotential for regeneration of shoot buds. The results suggest thatH^sub 2^O^sub 2^ is directly correlated with the morphogeneticprocess in strawberry callus (Tian et al. 2003). Types II and IIIstrawberry calli, which showed higher regeneration capacity, hadfive- and ninefold higher contents of intracellular H^sub 2^O^sub2^, and three- and fourfold higher contents of intracellular O^sup- ^^sub 2^ compared to type I callus, which exhibited a much lowerregeneration capacity (Tian et al. 2004). Types II and III callialso showed much higher activities of antioxidant enzymes than typeI calli. The results indicated that ROS might play a dual role inthe regeneration of strawberry calli. On one hand, a certain levelof a typical ROS may have a positive effect on strawberryregeneration. On the other hand, high levels of another ROS areinhibitory 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 itinhibited shoot organogenesis (Dutta and Datta 2003).

Antioxidants and the control of tissue browning and necrosis inplant tissue culture. Numerous studies reported tissue browning/necrosis leading to poor plant regeneration in vitro and successfuluse of antioxidants solving the problems in the tissue culturehistory (Table 1). Most of the studies were associated withdicotyledonary plant species; however, several antioxidants such asascorbate, cysteine, dithiothreitol (DTT), glutathione, andtocopherol were also successfully used in monocotyledonary plantspecies (Table 1). Among the studies, four of them had been welldocumented. Ziv and Halevy (1983) reported that using anantioxidant, DTT, controlled oxidative browning of tissues duringin vitro propagation of Strelitzia reginae. Terminal and axillarybuds, which were treated with a solution containing 0.04% DTT bysubmersing the buds for 24 h and then culturing the buds on an agarmedium with charcoal or by culturing the buds on paper bridges in aliquid medium with 0.04% DTT, were able to develop to shoots. Alsothe degree of oxidative browning of the shoot tip expiants wasreduced from a rating of 4 (without DTT) to a rating of 1.5. Shoottips of apple (Malus pumild) rootstock M.26 turned brownimmediately after being excised from expiants during in vitropropagation. The browned shoot tips would neither proliferate nordevelop into plantlets (Nomura et al. 1998). Glutathione (GSH) wasapplied before the propagation to prevent browning of the shoot tipexplants (Nomura et al. 1998). Shoot development from the explants,which were treated with 0.1 mM GSH solution by dipping them in thesolution prior to culture, was compared with that of the explantswithout GSH treatment (control). In the GSH treatment, 100% ofshoot tips developed into normal shoots after 120 d, whereas theresult from the control was 40%. The results showed thatapplication of GSH prior to the culture promoted the normaldevelopment of shoot tips. Apparently the major effect of GSH onthe shoot tip development was the protection from browning of theshoot tips. For in vitro propagation of Protea cynaroides,oxidative browning of shoot segment expiants is a major problem (Wuand Toit 2004). However, almost all expiants, which were immersedin a solution containing 100 mg/l ascorbic acid and 1,500 mg/lcitric acid for 1 h, and grown under 16 h photoperiod, had a 100%of the expiants survived and developed shoots, while only 20% ofthe expiants survived without the antioxidant treatment (Wu andToit 2004). Adonis amurensis is a perennial ornamental plant whoseshoot tip expiant darkening is a major obstacle to establish invitro propagation (Park et al. 2006). Normally, about 20% of theexpiants survive the initial stages of the culture (Park et al.2006). However, when they were soaked in an antioxidant solutioncontaining 300 mg/l ascorbic acid and 300 mg/l citric acid for 30min prior to the culture, survival rate was about two times higher(53.3%) than the nontreated control (23.0%) (Park et al. 2006).

Perl et al. (1996) found that short exposures of embryogenic calliof Vitis vinifera cv. Superior seedless to a diluted solution ofAgrobacterium resulted in tissue necrosis. The necrosis seemed tobe oxygen-dependent and correlated with elevated levels ofperoxides. Therefore, the inclusion of both 1% PVPP and 2 mg/l DTTin coculture medium was found to improve expiant viability duringand after cocultivation. They observed that the necrosis of theembryogenic calli was completely inhibited by these antioxidantswhile Agrobacterium virulence was not affected. These antioxidantsenabled the recovery of stable transgenic grape plants resistant tohygromycin. In another study Das et al. (2002) successfully used 1%PVPP and 2 mg/l DTT to control browning and necrosis in grape leaftissue expiants in a coculture medium during Agrobacterium-mediatedtransformation.

It was found that the coculture of sugarcane leaf spindle sectionswith A. tumefaciens induced a rapid necrosis of the tissue(Enriquez-Obregon et al. 1997, 1998; Gustave et al. 1998). Tominimize the necrosis, the leaf spindle expiants were incubated ina 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 inoculationwith Agrobacterium and then cocultured in a cocultivation mediumwith the same antioxidants at the same concentrations,respectively, after the inoculation with Agrobacterium. By doingso, the percentage of the expiant viability was increased from 10%(without the antioxidants) to 90%. In addition, the percentage ofGUS positive expiants was increased from 0% (without theantioxidants) to 100%. Enriquez-Obregon et al. (1999) alsoinvestigated the effects of the three compounds on the necrosis ofshoot meristem expiants prior to infection in rice transformation.The expiants, which were incubated in a liquid medium containing 20mg/1 ascorbic acid, 40 mg/1 cysteine, and 5 mg/1 silver nitrate for6 h in the dark, had an average of 6% of the each expiant areaproducing the necrosis, but the explants without the antioxidanttreatment had 80% of the each expiant area producing the necrosis.The antioxidant treatment increased rice transformation efficiencyfrom 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 regionby including 400 mg/l cysteine in cocultivation medium,subsequently, resulting in a twofold increase in transformationefficiency (2.1% with cysteine vs. 0.9% without cysteine).Browning/necrosis on the expiant tissues was also reduced. Thestudies also showed that the frequency of transformed cells wasincreased only when cysteine was present during cocultivation ofAgrobacterium and cotyledonary-node explants. Later, the authorsreported the cocultivation medium with 3.3 mM cysteine, plus 1 mMDTT 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). Applying400 mg/1 cysteine in the coculture medium increased both thefrequency of transient a-glucuronidase (GUS) expression in targetcells of com (56% with cysteine vs. 17% without cysteine) and thestable transformation frequency (6.2% with cysteine vs. 0.2%without cysteine) (Frame et al. 2002). However, cysteine reducedthe percentage of the immature zygotic embryos giving rise toembryogenic Type II callus from 99% when noninfected immaturezygotic embryos incubated on cocultivation medium without cysteineto 52% when the embryos cultured on cocultivation medium containingcysteine (Frame et al. 2002). Zeng et al. (2004) further confirmedthat the inclusion of 400 mg/l cysteine in a cocultivation mediumincreased stable transformation from 0.2% (without cysteine) to5.9% in soybean Agrobacterium-mediated transformation.

Adding glutathione to the selection medium reduced hyperhydricityof leaf expiants, increased leaf expiant viability, and increasedthe frequency of transformation from 13% (without glutathione) to45% 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, andglutathione in a cocultivation medium, on ROS production,antioxidant activity, and stable transformation efficiency duringpeanut Agrobacterium-mediated transformation. They found thatglutathione, tocopherol, and selenite not only eliminated theformation of H^sub 2^O^sub 2^ produced in wound tissue duringpreparation of leaf expiants and their cocultivation with A.tumefaciens, but also decreased malondialdehyde (MDA) formation andenhanced the activities of the antioxidant enzymes such assuperoxide dismutase (SOD) and catalase (CAT). The inclusion of 100mg/l glutathione, 50 mg/l tocopherol, and 20 mg/l selenite in thecocultivation medium increased the transformation frequencies from3.9% (no antioxidant) to 14.6%, 10.3%, and 12.4%, respectively(Zheng et al. 2005).

Lipoic acid (LA) is a sulfur-containing compound involved inseveral multienzyme complexes such as pyruvate dehydrogenase, ct-ketoglutarate dehydrogenase, branched-chain keto aciddehydrogenase, and glycine decarboxylase complex (Packer et al.1995). In animals, free LA and dihydrolipoic acid are metabolicantioxidants that are able to scavenge most reactive oxygenspecies, to recycle other antioxidants such as vitamin C,glutathione, and vitamin B, and to increase the expression of genesinvolved in the regulation of normal growth and metabolism as wellas redox regulation of gene transcription (Packer et al. 1995;Packer and Tritschler 1996; Packer et al. 1997). Therefore, LA wasinvestigated in Agrobacteriummediated transformation across fivedifferent plant species, and it has significantly improved thetransformation methods, even for recalcitrant genotypes (Dan et al.2004; Dan 2006). Frequencies of independent transgenic plant eventswere increased in soybean from 0.6% to 3.6%, in potato from 3% to19%, in tomato from 28% to 94%, and in wheat from 2.9% to 5.4%. Thefrequency of putative transgenic embryo was increased in cottonfrom 41% to 61%. The frequencies of shoot escapes were reduced insoybean from 92% to 72%, in potato from 50% to 16%, and in tomatofrom 91% to 53%, under the optimal conditions. This study alsodemonstrated that an increase of the transformation frequency andreduction of escapes in tomato were accompanied by a twofoldreduction in the severity of the browning/necrosis ofAgrobacterium-toansfomied tissues, a twofold increase in thesurvivability of the transformed tissues, a fourfold increase inthe percentage of transgenic shoots, and a threefold reduction ofthe percentage of shoot escapes when using LA under optimalconditions. The application of LA in plant transformation hasdramatically solved the three common problems in planttransformation: browning/necrosis of the transformed tissues,recalcitrance, and shoot escapes, which severely limit the numberof transgenic plants produced.

Analysis of published information indicates that the antioxidantsused in plant transformation can be classified into two groupsbased on their biological function. The first group, consisting ofascorbic acid, cysteine, DTT, lipoic acid, and PVPP, functions toreduce expiant necrosis, increase viability of expiants, andimprove transformation efficiency while the other one, whichinclude glutathione, selenite, and alpha-tocopherol, reduceshyperhydricity and ROS and increases transformation efficiency.

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 DNAfragmentation, 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 apoptosisin maize tissues. p35 is reported to act as a direct inhibitor ofcaspases whereas lap may act upstream to prevent activation ofcaspases. Caspases control most events of apoptosis in vertebratesand in invertebrates and are responsible, either directly orindirectly, for the cleavage of cellular proteins. The proteinsinclude nuclear proteins such as poly (ADP-ribose) polymerase, DNA-dependent protein kinase, and lamins, as well as actin (Nagata1997). The evidence that these genes could affect the reaction ofmaize cells to an apoptotic stimulus strongly indicates that plantmay have caspase-like proteases regulating apoptosis. A caspase-like proteolytic activity was also reported in tobacco plantsundergoing an HR triggered by an infection with Tobacco mosaicvirus (del Pozo and Lam 1998). Molecular identification of ROSsignaling during programmed cell death (PCD) in plant. One of thefirst genes identified and isolated in ROS-associated plant celldeath is LSDl that encodes a zinc-finger protein. This protein,together with two other zinc-finger proteins (LOLl and LOL2), couldact as a molecular rheostat, sensing changes in ROS homeostasis,thereby controlling apoptosis through the regulation of apoptoticgenes (Dietrich et al. 1997; Epple et al. 2003). The inactivationof the Arabidopsis Executerl gene completely abolished singletoxygeninduced cell death (Wagner et al. 2004). The chloroplasticprotein, which is encoded by Executerl, might perceive nonscavengedsinglet oxygen species within the chloroplast. Other importantstress signal transducers include mitogen-activated protein kinases(MAPKs) that act upstream of the oxidative burst during ozonetreatment and the HR (Ren et al. 2002; Samuel and Ellis 2002). Theprimary ROS activated tobacco MAPK is the salicylic acidinducedprotein kinase, which is required during harpindependent PCD(Samuel et al. 2005). A MAPKKK of alfalfe activates cell deathinduced by H^sub 2^O^sub 2^ through a specific MAPK-scaffoldingaction (Nakagami et al. 2004). A recent finding demonstrates theconserved Arabidopsis BCL2associated athanogene protein has beenshown to be induced by H^sub 2^O^sub 2^ and capable of provokingPCD in both yeast (Saccharomyces cerevisiae) and plants (Kang etal. 2006).

Mechanism to control programmed cell death associated with ROS inplant. Several recent studies have begun to unravel the possibleregulatory mechanisms of programmed cell death associated with ROSin plants (Breusegem and Dat 2006; Patel et al. 2006). The mostextensively studied form of plant PCD is HR to pathogen infection(Greenberg and Yao 2004; Soosaar et al. 2005). Recent evidenceindicates that autophagy, which is induced during the plant defenseresponse, is one mechanism by which HR-PCD is controlled (Liu etal. 2005). Autophagy is a process hi which cytosol and organellesare sequestered within doublemembrane vesicles that deliver thecontents to the lysosome/vacuole for degradation and recycling ofthe resulting macromolecules (Klionsky 2005). Several AuTophaGy(ATG) genes, which are required for autophagic activity, haveidentified via genetic screens in yeast (Levine and Klionsky 2004).The findings of Liu et al. (2005) imply that there is a prodeathsignal that moves out of the HR lesion into the surroundinguninfected tissue. Therefore, autophagy most likely alters theinduction, movement and/or recognition of the pro-PCD signal.Autophagy is not required for HR-PCD execution but is required tolimit PCD to the infection sites in plant and might prevent aprodeath signal from initiating PCD in healthy tissue (Liu et al.2005). Another study also suggests that autophagy has an anti-PCDfunction in innate immunity (Patel et al. 2006). However,examination of the function of ATG genes in other higher eukaryotesindicates that autophagy might have a dual role: pro- and/oranti-cell death (Yu et al. 2004; Boya et al. 2005; Pattingre et al.2005). The study from Torres and Dangl (2005) suggest that ROS andnitric oxide are possible candidates for a pro-PCD signal. Asextracellular ROS are produced before the onset of HR-PCD, ROS havebeen thought to function as pro- PCD signals, either by directlykilling the pathogen or by acting as signaling molecules thatmediate defense responses (Torres and Dangl 2005).

Apoptosis functions as an important defense strategy by host cellsagainst viral invasion. Many viruses contain the antiapoptoticgenes to block the defense-by-death response of host cells (Wang etal. 2004). The expression of antiapoptotic genes such as iap andp35 from baculoviruses, ced-9 from Caenorhabditis elegans and bcl-2from humans in tobacco, tomato, and passion fruit plant (Passifloraspp) suppressed the extensive cell death caused by fungal andbacterial pathogens and also enhanced resistance to some abioticstresses such as wounding, salt, cold, UV, and herbicides (Dickmanet al. 2001; Lincoln et al. 2002; Chen and Dickman 2004; Li andDickman 2004; Xu et al. 2004; Freitas et al. 2007). Particularly,baculovirus p35 and iap genes were expressed in com embryos andembryogenic calli and their expression reduced tissue browning 3 dafter cocultivation with A. tumefaciens (Hansen 2000). Theseantiapoptotic genes can be engineered to be expressed in plants forcontrolling tissue browning/ necrosis and enhancing planttransformation efficiency, especially in recalcitrant or poorlytransformable plant species.

Potential Mechanisms of Antioxidants for Improving PlantTransformation

Exposure of plant tissues to Agrobacterium during planttransformation leads to browning/necrosis of targeted cells/tissues, which affect transformation efficiency (Kuta and Tripathi2005). Browning/necrosis of targeted cells/tissues affects planttransformation in two ways. Browning/ necrosis may occur intransformed cells within expiant tissues, inhibiting regenerationof the transformed cells/ tissues. secondly, necrotic tissues areknown to accumulate antimicrobial substances (Goodman and Novacky1994), which may inhibit the potential of Agrobacterium to colonizeplant cells and transfer T-DNA. Thirdly, the active release ofchemical signals, which induce the vir genes in Agrobacterium,occurs only in living cells (Shaw et al. 1991). This also couldreduce the potential of Agrobacterium to transfer T-DNA intoplants. The inhibition of regeneration of transformed cells/tissuesmay promote growth of non-transformed cells/tissues even underselective pressures and, subsequently, result in production ofshoot escapes. With regard to Agrobaterium, the ROS, producedduring attempted transfection could be toxic enough to directlykill the attacking Agrobacterium (Wojtaszek 1997), therebysubsequently preventing Agrobacterium from colonizing plant cellsand transferring T-DNA into plants.

Perl et al. (1996) observed that elevated levels of peroxidaseactivity in grape tissues correlated with Agrobacterium-inducednecrosis in the host during Agrobacterium-mediated transformation.Peroxidase is known to mediate oxidative cross-linking ofstructural proteins in the cell wall (Somssich and Hahlbrock 1998),and the Agrobacterium-induced increase in peroxidase activity ingrape tissues could further confirm the role of oxidative burst inHR in plants to Agrobacterium infection. Deng et al. (1995)demonstrated that at least two genes residing within the T-DNAregion of Agrobacterium are responsible for inducing necrosis ingrape tissues. Furthermore, the aviR gene in Agrobacterium vitiswas found to be associated with Agrobacterium-induced HR (Zheng etal. 2003). aviR is homologous to luxR, which implies that the^groeactentww- induced HR is regulated by a quorum-sensingmechanism. The Agrobacterium-mdaced HR could lead to rapid andlarge generation of ROS in target plant cells, resulting in plantcells/tissues necrosis, oxidative stress on the invadingAgrobacterium cells, production of toxic antibacterial substances,and the deleterious effects on DNA molecules, especially at thesite of oxidative burst (Zheng et al. 2003). All of these factorscould significantly reduce the efficiency of stable transformationof plants.

Conclusion

Preliminary research on Agrobaterium-mduced plant cell death hasshown that Agrobacterium likely causes browning/necrosis oftransformed plant cells/tissues in vitro and ROS production duringAgrobacterium infection in vivo induces necrosis. The ROS couldkill the attacking Agrobacterium, thereby preventing Agrobacteriumfrom infecting plant cells/tissues and delivering T-DNA intoplants. Also, the necrosis prevents regeneration of transformedcells/ tissues.

Using antioxidants in plant tissue culture and transformationreduces the browning/necrosis of nontransformed and transformedcells/tissues and the frequencies of shoot escapes. In addition,the antioxidants increase the stable transformation efficienciesacross dicotyledonary and monocotyledonay plant species, indicatingtheir potential roles of controlling ROS for efficient productionof transgenic plants.

Advancing Agrobacterium-mediated transformation technology requiresan understanding of the mechanisms of Agrobacterium- inducedbrowning/necrosis and the roles of antioxidants in facilitatingplant transformation. The functions of the antioxidants describedabove have raised important questions. Do ROS produce whenAgrobacterium 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 ROSthrough regulating the expression of genes involving ROS-generatingsystems such as NADPH oxidase or pH-dependent cell wall peroxidase?Do the ROS scavenging and/or down-regulating activities prevent thebrowning/necrosis of transformed cells/tissues? Or do theantioxidants alter the expression of genes that play an importantrole in the regeneration of transformed cells/ tissues byregulating the production of ROS that damage cells/tissues, or byactivating other metabolic pathways? Once these questions areaddressed, we can control apoptotic responses during wounding andAgrobacterium infection in plant transformation more effectively.This will undoubtedly lead to the development of more efficientplant 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 / Publishedonline: 18 March 2008 / Editor: P. Lakshmanan (c) The Society forIn Vitro Biology 2008

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CONTACT:
Y. Dan
Institute for Sustainable and Renewable Resources
Institute for Advanced Learning and Research
150 Slayton Avenue,
Danville, VA 24540, USA
e-mail: Yinghui.dan@ialr.org

Y. Dan
Department of Horticulture and Forestry
Virginia Polytechnic Institute and State University
Blacksburg, VA 24061, USA


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