Indian Journal of BiotechnologyVol 2, January 2003, pp 17-25

Hybrid Necrosis in \Vheat-A Genetic System Showing Reduced Capacity toDetoxify Reactive Oxygen Species Leading to Programmed Cell Death

Geetanjali Sharma, B Srivalli and Renu Khanna-Chopra+Stress Physiology Laboratory, Water Technology Centre, Indian Agricultural Research Institute, New Delhi 110012, India

Hybrid necrosis in wheat is the premature gradual death of leaves and leaf sheath caused by two complementarygenes NeJ and Ne2 when brought together in a hybrid combination. Many promising wheat varieties are carriers ofthese genes, which limit the parental choice for transfer of desirable traits as the necrotic plants die withoutproducing seeds. The leaves of Kalyansona x C306 and WL711 x C306 hybrids showed enhanced generation ofsuperoxide radical and H202 than parents before and during progression of necrosis. Higher lipid peroxidation wasan early event in hybrid necrosis and was accompanied by a loss in membrane permeability and cell viability. Theanti-oxidant defense in necrotic hybrids was not well coordinated culminating in a persistent oxidative stress in theleaf especialiy in the chloroplast. The hybrid necrosis barrier was overcome in some crosses by culturing ears inmedium containing antioxidants and F2 seeds have been obtained. Hybrid necrosis is a unique genetic system forstudying the molecular mechanism of programmed cell death in plants. The cloning and characterization of Nel andNe2 may provide a tool having wide applications in agriculture. The review compares hybrid necrosis with otherPCD phenomenon in plants.

Keywords: hybrid necrosis, programmed cell death, oxidative stress, wheat

IntroductionHybrid necrosis is the premature gradual death of

leaves and leaf sheath (Fig. lA) caused by twocomplementary genes Nel and Ne2 when broughttogether in a hybrid combination (Hermsen, 1963).Many promising wheat varieties are carriers of Ne1 orNe2 genes located on chromosomes 5BL and 2BS,respectively (Nishikawa et al, 1974). This limits theparental choice for transfer of desirable traits atintraspecific i.e. among hexaploids as well asinterspecific level (hexaploids x tetraploids). Theseverity of necrosis varies greatly due to multipleallelism of these necrotic genes. In certain crosses, thePI shows severe necrosis and dies without producingseeds. Necrosis in the leaves indicates localized deathof the cells or tissue (Fig. 1B). Thus, hybrid necrosisappears to be a genetically regulated PCD process.

PCD is a process wherein there is a destruction of

*Author for correspondence:Tel: 25756012, 25742830; Fax: 91-011-25756012/25742830E-mail: rkc20@hotmail.com;renu_wtc@rediffmail.comAbbreviations:APX: Ascorbate peroxidase; CAT: catalase; DAS: days aftersowing; GR: glutathione reductase; H202: hydrogen peroxide;NADH: nicotinamide adenine dinucleotide (reduced); NADPH:nicotinamide adenine dinucleotide phosphate (reduced); POX:peroxidase; PCD: programmed cell death; ROS: reactive oxygenspecies; SA: salicylic acid; SOD: superoxide dismutase; TIC:2.3,5-triphenyl tetrazolium chloride.

unwanted cells through the activation of a geneticallycontrolled cell suicide machinery (Ameisen, 1996).The term programmed cell death. is sometimes usedsynonymously with 'apoptosis.' Apoptosis is a strictlyregulated process and is responsible for the orderedremoval of superfluous, aged or damaged cells. Thisform of PCD occurs as part of normal developmentalprocesses and differs from those which occur at thesite of environmental stresses such as pathogeninfection or physical wounding or in response to lowconcentration of toxins. These types of cell deathappear to occur through a controlled disassembly ofthe cell (Buckner et al, 1998). The other mode of celldeath i.c. necrosis is characterized by cellular swelling(oncosis) and lysis and is the outcome of severe andacute injury such as sudden shortage of nutrients,exposure to a high concentration of a toxin, heating orfreezing, etc (Cohen, 1993).

PCD in animal systems is a much researched andreviewed subject (Kroerner es al, l 998). Classicalexperiments have been conducted on Caenorhabditiselegans on the induction and action of specific genesthat bring about the controlled disassembly of the cell(Wadewitz & Lockshin, 1988). The regulation ofPCD is conserved in diverse organisms. In plants, theresearch in PCD has been the focus only in recentyears (Lam et al, 1999; Pennell & Lamb, 1997;Richberg et al, 1998). It has been suggested that

18 INDIAN J BIOTECHNOL, JANUARY 2003

C360 F1 WL 711

WL 711xC306

Fig. 1- (A) Necrotic wheat hybrid C306 x WL 711 and its parents,C306 and WL 711 at 110 days after sowing; (B) Progressivenecrosis in the hybrid leaf from the tip to the base.

despite some differences both plants and animalscould share some common components of a coremechanism to carry out PCD (Danon et al, 2000).Understanding the regulation of PCD in plants mayhave important applications in agriculture and post-harvest industries helping in prolonging the shelf-lifeof crops, fruits and vegetables. Also the suppressionof PCD could help in minimizing disease symptomscaused by pathogens.

ROS such as superoxide, H202 and hydroxylradicals are key players involved in signaling of PCD(Jabs, 1999; Lamb & Dixon, 1997; VanCamp et al,1998). Transgenic tobacco plants with reducedactivities of H202 scavenging enzymes such as APXand CAT had higher rates of PCD following bacterialinfection than wild type plants (Chamnongpol et al,1996; Mittler et al, 1999). Low doses of ROS inducesanti-oxidant enzymes, however, when theconcentration of ROS reaches a certain threshold, asignal transduction pathway that results in PCD isactivated (Levine et al, 1996). Thus, the survival ofplants depends on the coordinated effort of the anti-oxidant defense system. Oxidative stress could be acommon mechanism involved in PCD of plants. Adetailed analysis of the mechanism of hybrid necrosisis required to understand this process of cell death inplants as well as to overcome this barrier to genetransfer in wheat.

This review focuses on the phenomenon of hybridnecrosis as a type of PCD and the different processesassociated with PCD.

Hybrid NecrosisHybrid necrosis is common in wheat as the carriers

of the genes Ne1 and Ne2 are present in most of thepromising varieties of Triticum aestivum (Linn.).Depending on the nature of the cross, the first necroticsymptoms may appear at any growth stage of theplant from the 2nd leaf onwards. They first appear onthe leaves followed by the leaf sheaths. The stems andthe ears mostly remain normal green when the leavesand sheaths are already necrotic. In most crosses, thesymptoms progress from the tip of the leaf to its baseand from the older to the younger leaves. This is

\called 'progressive necrosis' (Fig. IE).

The degree of necrosis varies and nine necrosisgrades have been distinguished depending on theexpressivity of the Nel and Ne2 alleles. These allelescould be weak, moderate or strong and theirexpression is influenced by the environment(Hermsen, 1963). When drought tolerant wheatvariety C306 was crossed with high yielding varietiessuch as Kalyansona, WL711, 124, etc of Mexicanorigin, the FI progeny exhibited hybrid necrosis andthe plants died at various stages of developmentwithout producing viable seeds (Khanna-Chopra &PatiI, 2002). Kalyansona x C306 hybrid was severelynecrotic and died at 6-8 leaf stage while WL711 xC306 hybrid was comparatively less severe andsurvived up to flag leaf emergence stage (Table 1).The 124 x C306 hybrid survived up to ear emergencestage and exhibited leaf senescence followed bydeath. Thus, cv. C306 and Kalyansona are carriers ofstrong Nel and Ne2 alleles, while WL 711 and 124 arecarriers of moderate-strong and moderate Ne2 alleles,respectively. The interaction of strong Nel allele withstrong or moderate-strong Ne2 alleles produces wheathybrids, which die without producing grains.

Oxidative Stress and Hybrid NecrosisGeneration of ROS has been implicated in various

processes of PCD (Inze & Van Montagu, 1995). Thisincrease in ROS leads to lipid peroxidation, whichultimately causes membrane damage and finally leadsto cell death. ROS scavenging depends on thedetoxification mechanism provided by an integratedsystem of non-enzymic molecules like ascorbate,glutathione, carotenoids, a-tocopherols andflavanoids as well as enzymatic anti-oxidants. Theseenzymes include SOD (EC 1.15.1.1), which reactswith superoxide radicals and converts them to 0" andi-h02. H202 is then detoxified by CAT (EC 1.1 1.1.6)and/or APX (EC 1.11.1.11). Glutathione, an

SHARMA et al: HYBRID NECROSIS-A PCD PHENOMENON 19

Table l---Characteristics of necrotic wheat hybrids, derived by crossing drought tolerant cv. C306 (Nelcarrier) with high yielding wheat cultivars Kalyansona, WL711 or J24 (Ne2 carriers) and their parents(Khanna-Chopra & Patil, 2002)

Genotype Total leaf area (cm/) Maximum Tiller No./Plant Survival Stage(at 60 DAS) height (ern) (maturity)

Kalyansona x C306 43.2±4.7 21.6 3.4 VegetativeWL711 x C306 64.2±3.0 45.2 4.0 Flag leaf emergedJ24 x C306 257.1±1O.0 54.8 8.3 Ear emergedKalyansona 140.8 85.0 22.8 MaturityWL711 127.3 92.7 12.9 MaturityJ24 138.5 108.0 12.2 MaturityC306 112.3 120.0 21.2 Maturity

intermediary redox metabolite in the ascorbate-glutathione cycle of scavenging H202 is maintained inthe reduced state by GR (EC 1.6.4.2). In addition,non-specific (guaiacol) POX (EC 1.11.1.7) also playsan important role in the anti-oxidative protection.

Our studies showed that ROS were involved inmediating the cell death observed during hybridnecrosis in wheat. An increase in superoxide radicalswas observed even before the onset of necrosis in thehybrid leaves as compared to parents. The gradient ofsuperoxide levels from tip to the base was parallelwith the progression of necrosis (Fig. 2A) (Khanna-Chopra et aI, 1998). In 'lesion stimulating diseaseresistance response' mutant ilsd.I) of Arabidopsis,superoxide radical initiates a runaway cell deathphenotype (Jabs et al, 1996). Membrane boundNADPH-oxidase is known to be a source ofsuperoxide radicals during oxidative burst (Lamb &Dixon, 1997). During hybrid necrosis the activity ofNADPH-oxidase increased significantly only after50% necrosis in the hybrid leaf (Fig. 2B). Thus,NADPH oxidase may not be responsible for theincrease in superoxide radicals preceding onset ofnecrosis. A higher H202 content was observed evenbefore the onset of necrosis in the hybrid leaves ascompared to the parents and increased several foldwith the progression of necrosis (Fig. 3A). Thus, theearly on higher ROS generation in the hybrid leafmediated cell death.

Lipid peroxidation and membrane permeabilitywere higher in hybrid leaves than the parentsthroughout the ontogeny (Fig. 2C) (Dalal & Khanna-Chopra, 1999). Cell viability measured by TICreduction showed significant correlation withconductivity. Increase in conductivity and decrease inTTC reduction was observed with progression ofnecrosis (Fig. 20). Thus, in necrotic leaves of hybridwheat, a close relationship was observed between

lipid peroxidation, membrane permeability and cellviability. Loss in chlorophyll and carotenoids wasobserved only after 50% necrosis stage. Similarly, thephotosynthetic efficiency, measured as Fv/Fm,declined only after the appearance of visible necrosis(Dalal & Khanna-Chopra, 1999). The resultssuggested that increased superoxide radical mighthave induced peroxidation of membrane lipids furtherleading to more generation of free radicals. Thesereactions being autocatalytic and non-reversible innature continued and caused membrane damage thusdisturbing the homeostasis required for the normalfunctioning culminating in the death of cells. Inspiteof higher lipid peroxidation and membrane damagedelayed effect on photosynthetic pigments and PSIImight be due to differential sensitivity of chloroplastmembrane and PSII to superoxide radical (Shen et al,1997).

3000....--------, ~. C' 14 ....-------...,

.8:::UB~.~ SiJ 1~ _5 0.8.~ .5 0.6;t: = 04p.....:A ~ 0.2~ ~ 0 L-.,.,- __ --,-__ .....J

0% 25'% 50% >50%

Stages of necrosts

" 2500 A~Q~.§ ~ 2000~ = 1500'~ ~ 1000b '"go g 500Vl 0

4L -':"'6 -";"8--10--12---"

Da)'s after leaf emergence0.2....--------, _ 300,--------...,

!. 250 D=200 ~~~ 150 •••I- 100

~ 50~ L- ~_~_~00 20 40 60 80

Conducriviry (0-iJ)

cg 0,15'".g~"P.;;' 0.1

~ ~J

'" -005]. ~'....l ~ 0 L..--""--""'-- ....•....-'---'--..J

4 6 8 10 12 14 16Days after Itaf emergence

Fig. 2-Changes in (A) superoxide anion, (B) NADPH oxidase and(C) lipid peroxidation measured as MDA content and (D)correlation between conductivity and TIC reduction by the hybridcells at different stages of leaf development in necrotic wheathybrid WL711 x C306 (.) and its parents WL711 (e) and C306("').Vertical bars indicate SE (n=3). In some cases error bars aresmaller than the symbols.

20 INDIAN J BIOTECHNOL, JANUARY 2003

Study of the anti-oxidant enzyme system in thehybrid leaves at different developmental stagesrevealed a differential response in necrotic wheat

.hybrids as compared to their parents (Dalal &Khanna-Chopra, 2001). Kalyansona x C306 hybridexhibited more severe necrosis than WL711 x C306.In Kalyansona x C306 hybrid. SOD activity showedno increase over the parents whereas WL 711 x C306hybrid showed an early increase, but it was possiblyinsufficient to scavenge increased superoxide.Activities of APX, GR and POX were enhanced,whereas CAT exhibited a decrease in activity with theappearance of visible necrosis in both the hybrids.The isozyme profile of the anti-oxidant enzymes wassimilar in the hybrids and their parents. One existingisoform of POX showed an early appearance in the

. hybrid and increased in intensity with the progressionof necrosis thus suggesting a possibility of POX as a

5~------'------,

3

source of H202. Peroxidases can generate H202 byoxidation of NADH, NADPH, thiols and certainphenols (Pichorner et al, 1992). Thus in necroticwheat hybrids, the anti-oxidant management was notwell co-ordinated leading to a persistent oxidativestress, which caused cell death. The response ofnecrotic wheat hybrids differed i'1 magnitude atdifferent developmental stages of the leaves, whichmay be related to the intensity of necrosis expressedby the hybrids.

Subcellular compartmentation of anti-oxidants isnecessary for the efficient quenching of ROS at theirproduction sites as chloroplasts, mitochondria andperoxisornes are intracellular generators of ROS. Inplants, chloroplasts are a major site of ROSproduction during photosynthesis (Asada, 1994) .Since necrotic lesions appeared mainly on the leaf and

:::' 200 I---' 180~ 'E 160'-' 0 1~01: a 120~ Oil 100g S 80'"' 0 60

SHARMA et al: HYBRID NECROSIS-A PCD PHENOMENON

leaf sheaths, the anti-oxidant defense in thechloroplasts was investigated. Low ascorbate andglutathione levels were detected in the chloroplasts ofthe hybrid before the onset of necrosis (Fig. 3 B & C).Chloroplastic SOD activity also followed a similartrend (Fig. 3 D & E). Thus hybrid necrotic leavesexhibited ineffective management of oxidative stressin chloroplasts culminating in cell death. Based onthese results a hypothetical model for the involvementof oxidative stress during hybrid necrosis is proposed(Fig. 4). According to the model, enhanced ROSpreceding necrosis led to higher lipid peroxidation,loss of membrane permeability and cell viability.Anti-oxidant defense system in the chloroplastscomprising the metabolites, ascorbate and glutathioneand enzymes SOD, APX and GR exhibited loweractivity even before the onset of necrosis in hybridleaves as compared to parents. Lack of co-ordinationin the anti-oxidant enzyme activities coupled withdecline in CAT activity resulted in an increase insuperoxide and H202 in the cell. Thus, increased ROSalong with the inability ~o manage the oxidative stresssignalled PCD resulting in necrotic lesions.

Generation of ROS is a light dependentphenomenon (Foyer et al, 1994). The longevity of theseverely necrotic Kalyansona x C 306 hybrid wasincreased by manipulating the light intensity,temperature and photoperiod in the controlledenvironment of phytotron. Some hybrids reached flagleaf emergence stage and very few showed earemergence and grain development (Sharma &Khanna-Chopra, 2001).

Processes Associated with Other Plant peDPhenomena

Signaling processes that activate PCD in plants areas diverse as in animals. Besides ROS, a requirementof calcium signaling has been implicated in thehypersensitive reaction (HR) induced cell death usingcalcium channel blockers (Pennell & Lamb, 1997).Another positive feedback regulator of cell deathduring HR is salicylic acid (SA) (Richberg et al,1998). The conversion of SA into catechol by a geneencoding salicylate hydroxylase (NahG) couldsuppress HR cell death in different Arabidopsismutants (Shah et al, 1999). SA has been implicated inozone-induced cell death as well (Rao & Davis,1999).

Hormonal balance (auxins and cytokinins) is acritical signaling component in the differentiation ofZinnia tracheary elements (Fukuda, 1996). Arequirement of ethylene is necessary in the cell death

21

caused by a fungal toxin 'victorin' (Navarre &Wolpert, 1999). In cell death of barley aleuroneprotoplasts, giberellic acid (GA) stimulates thesecretion of hydrolytic enzymes and triggers the onsetof PCD, whereas abscisic acid antagonizes the effectsof GA and inhibits PCD (Bethke & Jones, 200 I).

One of the simplest hallmarks of PCD in plants isthe DNA ladder. A DNA ladder is the product ofchromatin digestion by nuclease(s) without theconcurrent protease activities on histone (Wylie.1980). Thus, a DNA ladder suggests that cellorganelles such as vacuoles or lysosomes arepreserved to some extent during cell death process.DNA ladder can be detected using a terminaldeoxynucleotidyl transferase-mediated dUTP nickend labelling (TUNEL) reaction. In plants, DNAladder has been reported in several conditions such assenescence (Yen & Yang, 1998), endospermdevelopment (Young et aI, 1997), UV irradiation(Danon et aI, 2000), death induced by pathogen toxin(Navarre & Wolpert, 1999)~ nutrient deprivation(Callard et aI, 1996), etc.

Plants undergoing PCD show cytoplasmicshrinkage and condensation but no apoptotic bodiesare formed (Lam et aI, 1999). This is due to theabsence of phagocytosis by the neighbouring cells ormacrophages, which engulf the cell corpses due to thepresence of a rigid cell wall in plants. Thus, thedegenerated cytoplasm and nucleus in plant cells maybe eliminated by other processes like vacuolarautophagy, etc.

The importance of caspases for apoptosis has beenevidenced by the fact that caspase activationcorrelates with the onset of apoptosis. These proteasesonce activated by apoptotic signal, systematicallydismantle and package the cell by cleaving keycellular proteins (Wolf & Green, 1999). In plants.caspase- like proteolytic activities have been observedduring HR induced cell death (del Pozo & Lam,1998). Peptide inhibitors of caspases could block theHR, whereas classical protease inhibitors could notsuppress it. Tobacco transgenic plants encoding Ced-9(a pro-survival cell death regulator) could delay HRinduced cell death and death caused by U V-Bandparaquat (Mitsuhara et al, 1999). In addition tocaspases, other cysteine proteases have beenimplicated as having a role in plant PCD (Solomon erat, 1999; Xu & Chye, 1999). Involvement of anextracellular serine protease has also been shown toregulate cell death activation during TE

22 INDIAN J BIOTECHNOL, JANUARY 2003

CELL WALL required (Lancomme & Santa Cruz, 1999).PLASMA MEMBRANE

vHP°2 /'1 (LIpid ptro:xldatiOIl ••r~Mtlllbrane damage) /" IOR (Ddayed Illa'ellSe)

i .> I CATi0i-----------. HzOz--_.:!.I tSOD ---. HzO •(InmIl1dmt incruu) L iROS __ Necrotic It$lom

~(LIpid pU'oxldatlollMtlllbNlllt damage)

Fig. 4-A proposed hypothetical model to explain the involvementof oxidative stress in the mechanism of hybrid necrosis. Figurenot drawn to scale. The dashed arrows indicate impairedpathways. APX, ascorbate peroxidase; AsA, Ascorbic acid(reduced); CAT, catalase; GR, glutathione reductase; GSH,glutathione (reduced); H20, water; H202, hydrogen peroxide; O2,molecolar oxygen; O2', superoxide radical, PSI, photosystem I;PSI!. photosystem II, ROS. reactive oxygen species, SOD,superoxide dismutase.

differentiation (Groover & Jones, 1999). However,substrates for some of them are not known as they aredifferentially inhibited. Thus, proteases with differentspecificities may regulate cell death in differentspecies. In animals, other processes include thecleavage of poly (ADP-ribose) polymerase, the firsttarget proteins to be specifically cleaved by caspasesinto a 89 kDa apoptotic fragment (Lazebnik et al,1994). However, abundance of poly (ADP-ribose)polymerase is generally very limited in plants(Krishnamurthy et al, 2000).

The mitochondrion has emerged as a conserved sitewhere cell death signals in the form of ROS andprotein factors can be generated. The release of suchfactors like cytochrome C activates caspases intostarting a cascade effect of activations of othercaspases family members in animals. A specificrelease of cytochrome C from intact mitochondria hasbeen shown in cucumber cotyledon undergoing PCD(Balk et al, 1999) and menadione induced cell death(Sun et al, 1999). Bcl-2 related proteins can eitheractivate or repress the leakage of cytochrome Cthrough the membrane of the mitochondria (Shimizuet al, 1999). The gene encoding the pro-apoptoticregulator Bax from mammalian systems whenexpressed in tobacco was found to activate HR-likecell death and its localization in mitochondria was

Mutants Showing Different Types of PCDCortical Cell Death Mutants in Cereal Roots

Cereals show increasing root cortical cell death(RCD) from the tip and upwards. RCD is likely toplay an important role in fungal establishment. Deadcells may serve as a food base for initial colonizationof the fungi, which subsequently kill others by toxinproduction. Certain root necrosis mutants have beenobtained in soybean (rn mutants). These roots showinduction' of peroxidases and are more resistant topathogen infection by fungal spores of Phytophthorasojae. Structure of dying cells in rn roots shows the

, hallmark events of apoptosis and surprisingly, thesecells also exhibited classical features of necrosis. Thetwo morphologies may represent different stages of acommon pathway for PCD in plant roots, or twoseparate pathways could be involved. It has beensuggested that the rn gene could either negativelyregulate cell death or may be required for cortical cellsurvival (Kosslak et al, 1997).

Hypersensitive Response (HR)The molecular mechanism for HR involves

generation of oxidative burst in infected cells andother events of PCD. Certain genetic mutations areknown in maize, barley and Arabidopsis that result inthe development of lesions resembling the HR orother disease symptoms in the absence of pathogeninfection. In maize, locus rp 1 confers resistance to themaize common rust, Puccinia sorghi (Buckner et al,1998). Mutations in this locus cause cell deathwithout any infection from outside. The lethal leafspot (lls 1) mutant of maize is a propogative diseaselesion mimic. Homozygous plants for recessive allelesshow small chlorotic lesions at 2-4th leaf stage,proceeding from older to younger leaves. Mutantphenotype here is dependent on light and chloroplasts.Bacterial mutants having a mutation in the hrp locus,which is important for the secretion of virulence andavirulence factors, were developed. On using this lirpstrain, oxidative burst was detected but cell death didnot occur. So, this strain uncoupled the oxidativeburst from the cell death. Role of superoxide inexecuting HR has been studied using the recentlydeveloped Zsd (lesion stimulating disease) mutant ofArabidopsis, which shows an apparent HR aftershifting uninfected plants from short-day to long-daygrowth conditions and accumulates superoxide in leaftissues (Jabs et al, 1996). Another lesion mimic

SHARMA et al: HYBRID NECROSIS-A PCD PHENOMENON 23

Table 2-Plant system and lesion mimic transgenes/mutants showing PCD

Gene Source Function Reference

A. Hybrid necrosis wheat Not known Hermsen, 1963l. NelNe2 (Accumulate ROS in

leaves)B. Mutantsl. lls Maize Suppressor of cell death Buckner et al, 19982. Isd] Arabidopsis Accumulate superoxide on Jabs et al, 1996

transfer from short-day tolong day conditions

3. Less22 Maize Inhibits porphyrin Hu et al, 1998pathway

C. Transgenicsl. Antisense CAT Tobacco Removal of ROS Chamnongpol et al, 19962. Antisense APX Tobacco Removal of ROS Orvar & Elli, 19973. Antisense PPO Arabidopsis Heme biosynthesis Molina et al, 1999

mutant, Less 22 in maize developed necrotic spots onthe leaves similar to HR lesions (Table 2). This geneencodes uroporphyrinogen, which producesintermediates for the biosynthesis of chlorophyll andheme-containing enzymes such as catalase. Onaccumulation in the cells, uroporphyrinogen canbecome reactive and may transfer its energy tooxygen, thereby forming ROS. These ROS may thenkill the cells either by apoptosis or by necrosisdirectly. The CAT levels also show a decline in thesemutants thereby further causing more severe oxidativestress (Hu et aZ, 1998).

SenescenceIt is the final phase in the death of the plant

involving active turnover and recapture of cellularmaterial for use in other organs. Increase in ethyleneand ROS is seen with the progression of senescence(Inze & Van Montagu, 1995). Mutant studies inArabidopsis have shown that a dominant mutation inthe ethylene responsive (ETR) gene can make itinsensitive to ethylene and block natural senescence.Certain senescence upregulated genes (SENUs) andsenescence associated genes (SAGs) encode forcysteine proteases, which may be involved in the PCDphenomenon (Pennell & Lamb, 1997).

Studies in oats revealed that. a .host selective toxin,victorin produced by Cochliobolus victoriaeproduced senescence-like symptoms. -In addition,DNA from victorin-treated leaves showed apronounced laddering effect (Navarre & Wolpert,1999). Thus, senescing parts of, a plant may undergocell death by a series of well-regulated events of anapoptotic pathway. .

ConclusionsStudies of PCD reveal some degree of conservation

amongst the various PCD processes in plants andanimals. Mitochondria are known to be important inexecution of animal PCD (Kroemer et al, 1998), butonly recently its role in plant PCD has been firmlyestablished. The role of mitochondria during synergidcell death has been highlighted by Christensen et a/(2002). Involvement of ROS signaling in variousplant and animal PCD is reported (Jacobsen, 1996).During hybrid necrosis also, higher superoxide levelsare observed even before appearance of visiblenecrosis on hybrid leaves (Khanna-Chopra et aI,1998). Hybrid necrosis barrier in some wheat crosseswas overcome by culturing the ears before anthesis inmedium containing anti-oxidants and F2 seeds were'obtained (Dalal et al, 1999). Currently effort is beingmade to examine the molecular changes preceding-isible necrosis in wheat cross Kalyansona x C306

using subtractive hybridization. A necrotic hybrid(J24 x C306) showed apparent senescence therebyindicating that senescence and necrosis may share acommon pathway. Thus, necrotic wheat hybridsprovide a unique genetic system to study themechanism of programmed cell death in plants usingmodern tools of biotechnology. The following areasof research need attention: (l) the source of ROSgeneration, (2) identification of the occurrence ofvarious common processes and cellular changesinvolved in different types of PCD such as presenceof caspases, endonucleases and DNA ladder, etc.,(3) identification of the genes turned on before theonset or during the visualization of necrotic lesions

. and' compare them with the other plant genes cloned

24 INDIAN J BJOTECHNOL, JANUARY 2003

that regulate cell death e.g. lsdl gene, which encodesa zinc finger protein of a class known in animals toact as transription factors (Dietrich et al, 1997) and(4) to clone Nel and Ne2 genes, which may havewider applications in agriculture.

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